ErbB2 is a tyrosine kinase receptor for neuregulin, a molecule released by neurons as part of the intrafusal induction mechanism during differentiation Upon the binding of neuregulin to ERB2, the receptor becomes phosphorylated pErbB2 40 , leading to an increase in the transcription factor EGR3, which initiates the expression of genes for intrafusal differentiation Immunocytochemistry indicated positive identification of both bag and chain intrafusal fibers Fig. A sample image of a chain fiber is shown in panel A and bag fiber in panel B.
These interactions were quantified as Immunocytochemical analysis of intrafusal fibers and motoneurons co-cultures. There was found to be a 5. In order to determine if the innervations identified immunocytochemically were functional, patch-clamp electrophysiological recordings were performed on intrafusal fibers 15—30 days in vitro DIV.
Glutamate is an excitatory neurotransmitter that has been previously used to stimulate neurons in co-cultures with muscle without directly initiating myotube contraction The electrophysiological response to the addition of glutamate, both with and without MNs in the culture, was recorded from intrafusal fibers, identified by their morphological characteristics, in the co-culture Fig. Theoretically, the intrafusal fiber, if innervated, should be excited upon glutamate addition, while those not innervated should not be affected.
To confirm that the increased AP firing was initiated by ACh-mediated innervation, a blocking agent for ACh mediated synaptic transmission, curare, was applied after increased firing was initiated by glutamate treatment Immediate cessation of electrophysiological activity was observed Fig.
As a control, the same experiment was performed on intrafusal fibers in the absence of MNs where no significant change of activity was detected. The few intrafusal fibers not excited in the co-culture may not have been innervated, resulting in a lack of response to MN activity. Patch-clamp analysis of intrafusal fibers. A,B Representative bright field microscopy of patched intrafusal cells in motoneuron-muscle co-cultures A and muscle only controls B.
C,D Gap-free recordings from patched intrafusal fibers in motoneuron—muscle co-cultures C and muscle only controls D. Addition of glutamate marked with green arrows in the co-culture elicited increased activity and addition of curare marked with red arrow terminated activity C but no activity change was induced by either of them in intrafusal fibers in the muscle culture alone D.
Statistical analysis. Percentages of glutamate responding and glutamate nonresponding intrafusal fibers in muscle-motoneuron co-culture conditions and muscle only conditions. The recapitulation of the spindle fusimotor circuit in vitro with human cells provides a defined model to investigate the physiology of this circuit, which is otherwise extremely difficult to observe for human systems. The use of MNs and muscle derived from iPSCs also offers the possibility for their incorporation into human-on-a-chip systems designed for the investigation of patient specific neuromuscular diseases and deficits.
Here, a co-culture of human intrafusal fibers and human MNs was established to evaluate the interactions between these two important cell types from the sensory portion of the reflex arc. Initial morphological analysis via phase contrast microscopy indicated the two cell types were compatible in a serum-free defined system and formed close physical contacts suggesting synaptic interactions. Immunocytochemical analysis confirmed the identity of the cell types in these intercellular interactions.
The majority of intrafusal fibers in co-culture demonstrated increased excitation upon the application of glutamate 28 out of 34 , while only one intrafusal fiber in the monoculture responded. Furthermore, the glutamate-induced responses were terminated by the application of curare, a competitive antagonist of acetylcholine receptors. It should be pointed out that a single intrafusal fiber in monoculture conditions exhibited AP firing upon exposure to glutamate.
The presence of glutamate receptors on muscle fibers is known and has been researched 43 , Multiple mechanisms have been proposed regarding the function of glutamate receptors in muscle tissue, but none of which correspond to direct action potential generation 45 , 46 , 47 , 48 , 49 , Actually, a similar response was recorded from another intrafusal fiber in a muscle-only culture by adding media instead of glutamate data not shown , indicating the addition event itself could non-specifically induce muscle excitation, although very rarely.
To further confirm the muscle was not directly excited by glutamate in our system, glutamate dosage experiments data not shown were performed on muscle only cultures to evaluate the responsiveness of intrafusal fibers to increasing concentrations of glutamate. The lack of glutamate mediated electrical activity in these cultures, even at concentrations far higher than used in experiments reported here, demonstrates the lack of direct response to glutamate.
This evidence confirms that the single cell responsive to glutamate was not the result of a glutamate induced electrophysiological mechanism in this human model. Most existing in vitro systems used to study these diseases only investigate the motor perspective of neuromuscular interactions.
However, utilization and especially integration of sensory components is essential to investigate complicated neuromuscular diseases by the inclusion of afferent mechanisms. Additionally, the sensory portion of the reflex arc has been of interest to the field of pain research Drug discovery has been moving towards more repeatable and high-throughput organ-on-a-chip in vitro systems recently for use in preclinical compound evaluation to improve drug discovery efficiency 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8.
These systems provide a highly controllable and repeatable platform that can be tailored to more human-specific diseases by utilizing patient-derived iPSCs. Human intrafusal fibers were differentiated from human satellite cells provided as a gift from Dr.
Herman Vandenburgh. Biopsies were performed on adult volunteers according to procedures approved by the Institutional Clinical Review Board of the Miriam Hospital and were performed in accordance with the relevant guidelines and regulations. All samples, from the study participation and publication of images, were obtained with informed consent and de-identified before being sent to UCF. The differentiation protocol was adapted from our protocol for extrafusal fiber differentiation by inclusion of specific factors to facilitate intrafusal differentiation Specifically, thawed satellite cells from liquid nitrogen were plated onto glass coverslips at a density of cells per mm 2.
The cells were given a whole medium change of human skeletal growth medium every two days until confluent. At this point, the medium was switched to differentiation 2 medium described in detail in Guo et al.
The cells were fed every two days with differentiation 2 medium and maintained for four days. After this point, the medium was given a half change with NBActive4 differentiation medium.
The cells were maintained in this medium for the remainder of the culture and fed every two days until 15—30 DIV total when they were analyzed via electrophysiology or fixed for immunocytochemical analysis. Specifically, thawed SCSC cells 0. Upon confluence, the cells were trypsinized with 0. Cells were initially plated in priming medium and fed on day 2 of culture with a half change of medium. On day 4 the medium was half changed with human MN medium. The cells were maintained in the permanox dish for about 10 days.
The cells were plated into DETA coated glass coverslips for less than 10 days before being trypsinized as described above and replated onto either muscle cultures or control DETA coated glass coverslips. At this point the neurons either underwent exposure to intrafusal differentiation media co-cultures or were maintained in human MN medium monocultures. Cells were permeabilized with 0. Antibodies used and their concentrations are listed in Table 1.
Human motoneurons and human spinal cord stem cells were harvested for RNA extraction at less than 10 days in vitro. The patch-clamp recording chamber was filled with the same medium as utilized for cell culture. Action potentials were recorded in current-clamp mode under gap free conditions at zero holding potential. Before seals were established on the cells, offset potentials were nulled. Capacitance subtraction was used in all recordings.
All intrafusal fibers chosen in co-culture conditions were in proximity to MNs identified via morphological analysis. When excited, the MNs would excite innervated muscle fibers and the excitation was recorded. Once repetitive firing was consistent, curare was added in 30 ul doses of uM to the extracellular solution. The standard error of the mean for each proportion was calculated for the binomial distribution. Intrafusal fibers positive for BTX staining that had contact with neuronal processes positive for neurofilament that lead to an identifiable soma were counted.
Standard deviations were calculated and expressed as percent error. Sung, J. Microfabricated mammalian organ systems and their integration into models of whole animals and humans.
The former are attached to the Z-disc and the latter to the central M-band Fig. Furthermore, in collaboration with B.
Grove, further details regarding M-band proteins relationships to fibre type development in chicken Grove et al. By studying the M-band structure and composition of physiologically defined rat motor units in soleus, a slow muscle, and m. Essential parameters, such as contraction time, Z-disc width and mitochondrial content of fast and slow fibres were relative and varied between muscles, whereas the M-band structure overrode the intragroup variability in contraction times of slow and fast units within and between the two muscles Thornell et al.
Nevertheless, the M-band ultrastructure pattern of lines is not constant either in slow and fast fibres of different species, nor between different muscles of the same species. Consequently, as expected, the composition of the M-band also differs Thornell et al. Variations in M-line condition of bag 1 b1 and bag 2 b2 fibres in regions A, B and C of the poles of typical rat, rabbit and cat spindles.
From Barker et al. Color code: actin filaments, dark yellow; myosin filaments, blue; titin filaments, green. The transverse structures are the Z-disk black and the M-band red. The extra-sarcomeric filaments magenta are anchored to transmembrane proteins in the sarcolemma. The sarcomeric borders are delineated by the Z-discs Z in the middle of the I-band. Numbers give the distances in nm from the centre of the A-band, the M1-line. No detailed study on human limb muscle spindles where M-band structure has been related to M-band composition had been published until we presented the ultrastructure of three serially sectioned human lumbrical spindles Thornell et al.
The spindles were composed of two large bag fibres and varying numbers of chain fibres Fig. The latter fibres always contained a dense M-band and showed immunolabelling for myomesin and M-protein. One bag fibre, interpreted as bag 1 on the basis of double immunolabelling with anti M-protein and anti-slow-tonic MyHC, lacked M-bands in the equatorial and the main part of the polar regions.
In the other bag fibre, the lack of an M-band varied between spindles. In one spindle a short segment of the equatorial region lacked a dense M-band, in a second spindle M-bands were seen along the whole length of the bag fibre, whereas in the third the equatorial region and a small part of one of the poles lacked dense M-bands Fig.
This strongly argues for a lack of register of the thick filaments within the A-band. In the fibres with dense M-bands, the A—I-band borders were in perfect register [Fig. In negatively stained cryosections, where the proteins are embedded in the electron-dense stain, making the filaments appear in high contrast, there was a marked difference in appearance of the M-region between fibres showing no M-lines, 3 strong and 2 weaker or 5 equally strong lines Fig.
Schematic drawings depicting the organization of three serially sectioned human lumbrical spindles 1—3. Scale in millimeters, with centre at equator at zero. Two nuclear bag fibres with larger diameters together with variable numbers of nuclear chain fibres with small diameters were present in each spindle. Solid areas mark the regions of fibres where M-bands were present. Note the variable length of M-band absence inbetween the three bag 1 fibres of the different spindles.
Likewise, bag 2 fibres showed marked differences — one fibre had a short segment without M-band, the second spindle had a bag 2 fibre with presence of M-band along its whole length, whereas the third spindle had quite a long region showing no M-band. Electron microscopy of longitudinally sectioned intrafusal fibres of a human lumbrical muscle spindle.
The bag 1 fibre in 7 lacks a dense M-band and shows an irregular border between the A- and I-band. Longitudinally sectioned sarcomeres in ultrathin cryosections of intrafusal fibres from human lumbrical spindle. The sections are negatively stained with uranyl acetate, thereby proteins and filaments are seen in reversed contrast, i.
The densities of the three central lines are most distinct, however, at places an additional two lines can be recognized L Carlsson and LE Thornell, unpublished.
In parallel with the increased knowledge about the myofibrillar M-band, it became evident that muscle fibres, including intrafusal fibres Eriksson et al. Intermediate filaments, composed of desmin, interlinked the myofibrillar Z-disc in cross-register, and anchored them to the sarcolemma, nuclei and mitochondria.
Intra-sarcomeric filaments, composed of titin, spanned half a sarcomere from the Z-disc to the M-band. The molecular layout of defined domains of titin, myomesin and M-protein resulted in the first molecular model of the M-band Fig. Since then a tremendous amount of detailed information has accumulated regarding the molecular organization of the extra- and intra-sarcomeric cytoskeleton Fig.
Extended molecular models of the M-band have been proposed Fig. Furthermore, the realization that the M-band is elastic and serves a signalling function opens up also the possibility that M-band strain might also translate into modulation of metabolic activity, in addition to protein turnover and transcription regulation, and thereby regulate short-term adaptation of muscle to strain.
The intra-sarcomeric cytoskeleton of titin and its connection to the Z-disc, I-band and M-band is a hot spot of research, as seen from the number of publications and reviews recently published e. Unfortunately, studies on the extra- and intra-sarcomeric cytoskeletal organization in muscle spindle fibres are lagging behind. Extrapolating the impact of the M-band signals on extrafusal fibres and cardiomyocytes to muscle spindle function, the special organization of the M-band in bag and chain fibres and along the length of bag fibres must have significance for the function of the different types of intrafusal fibres.
At the moment one can only speculate how the detailed variations in MyHC expression in intrafusal fibres, in combination with M-band diversity in structure and composition, influence the special tasks of the muscle spindle. More precise information regarding the mechanism s ensuring the differential regulation of cytoskeletal proteins and myosin isoforms in the different regions and types of intrafusal fibres will serve as the backbone for further molecular modelling of neuronal interactions and mechanical factors for muscle spindle function.
Schematic presentation of the arrangement of titin, myomesin and M-protein in the M-band compatible with the immunoelectronmicroscopical results Obermann et al. Crosshatched and shaded boxes reflect Ig and Fn-domains, respectively. From Obermann et al. Overview of cytoskeletal structure-associated proteins at the M-band and the Z-disk: for further details, see Hoshijima Reproduced with permission.
Structures for several individual and multi-domain titin domains have also been solved A—, A—A, M1, M5. Although this model agrees with known interactions and ultrastructural locations of individual protein domains, alternative paths for titin are also compatible, and the conformation of large interdomain linkers represented as simple lines is yet unknown. Titin domains are shown in blue and are numbered; subunits of the myomesin dimer in shades of red.
Arrows denote the bipolarity of the myosin filament and point towards the antiparallel motor domain arrays in the A-band. From Gautel b with permission. However, one should bear in mind that for human muscles each has its own composition of muscle spindles, and each spindle has its special set of intrafusal fibres, indicating unique morphological and physiological characteristics. Structural complexity of the human muscle spindle system may fit well with its diverse functional roles in control of posture and locomotion, timing of locomotor phases, synergy formation, plasticity and motor learning, and to act as forward sensory models, i.
National Center for Biotechnology Information , U. Journal List J Anat v. J Anat. Published online Jul Author information Article notes Copyright and License information Disclaimer. E: es. Accepted Mar This article has been cited by other articles in PMC.
Keywords: cytoskeleton, M-band, M-protein, muscle spindle, myomesin, nuclear bag, nuclear chain, titin. Open in a separate window. Fig 1. Fig 2. Three intrafusal fibres In subsequent years, enzyme-histochemical and ultrastructural studies suggested two types of bag fibres, but the issue was controversial for many years see review by Boyd, Table 1 Classifications of intrafusal muscle fibre types compared with present classification.
Fig 3. Fig 4. Fig 5. Fig 6. Fig 7. Immunohistochemical evaluation of intrafusal type development in human muscle spindles In parallel to the progression of our immunohistochemical studies on human skeletal muscle development, where we could distinguish two generations of fibres early in development and the downregulation of embryonic and foetal MyHC upon fibre type specification of slow and fast MyHC-containing fibres Thornell et al.
Fig 8. Human intrafusal fibre types on basis of MyHC composition The summarized initial immunocytochemical analysis of MyHC isoforms in human spindles, in m. Revised fibre typing on basis of completion of the human genome project Eleven sarcomeric myosin heavy chain MYH genes were identified by the human genome project Fig.
Fig 9. Fig Molecular composition of the extra- and intra-sarcomeric cytoskeleton in relation to M-band function as a mechanical sensor with elastic properties In parallel with the increased knowledge about the myofibrillar M-band, it became evident that muscle fibres, including intrafusal fibres Eriksson et al.
Conflict of interest No conflict of interests to be declared. The M-band: an elastic web that crosslinks thick filaments in the center of the sarcomere. Trends Cell Biol. The molecular composition of the sarcomeric M-band correlates with muscle fiber type. Eur J Cell Biol. Frog muscle spindle at different functional conditions.
In: Kakulas BA, editor. Basic Research in Myology. Amsterdam: Excerpta Medica; The 2B myosin heavy chain gene is expressed in human skeletal muscle.
J Physiol. The muscle spindle. In: Engel AndrewE. New York: McGraw-Hill; Clara Franzini-Armstrong. Correlation between ultrastructure and histochemistry of mammalian intrafusal fibres. A study of mammalian intrafusal muscle fibres using a combined histochemical and ultrastructural technique.
Immunocytochemical characterisation of two generations of fibers during the development of the human quadriceps muscle. Mech Dev. Studies of the histochemistry, ultrastructure, motor innervation, and regeneration of mammalian intrafusal muscle fibres. Prog Brain Res. An immunofluorescence study. J Cell Biol. Most muscles contain both fast- and slow-twitch fibers, but in different proportions.
Thus, the white meat of a chicken, used to control the wings, is composed primarily of fast-twitch fibers, whereas the dark meat, used to maintain balance and posture, is composed primarily of slow-twitch fibers.
Upper trace of oscilloscope represents the action potentials of a descending pathway axon. With low rates of activity of the descending pathway, only small alpha motor neurons are activated, producing small amounts of muscle force lower trace of oscilloscope.
With increasing rates of descending pathway activity, intermediate-size alpha motor neurons are activated in addition to the small neurons. Because more motor units are activated, the muscle produces more force. Finally, with the highest rates of descending activity, the largest alpha motor neurons are recruited, producing maximal muscle force.
The motor system requires sensory input in order to function properly. In addition to sensory information about the external environment, the motor system also requires sensory information about the current state of the muscles and limbs themselves.
The muscle spindle signals the length of a muscle and changes in the length of a muscle. The Golgi tendon organ signals the amount of force being applied to a muscle. Muscle spindles are collections of specialized muscle fibers that are located within the muscle mass itself Figure 1. These fibers do not contribute significantly to the force generated by the muscle. Rather, they are specialized receptors that signal a the length and b the rate of change of length velocity of the muscle. Because of the fusiform shape of the muscle spindle, these fibers are referred to as intrafusal fibers.
The large majority of muscle fibers that allow the muscle to do work are termed extrafusal fibers. Each muscle contains many muscle spindles; muscles that are necessary for fine movements contain more spindles than muscles that are used for posture or coarse movements. There are 3 types of muscle spindle fibers, characterized by their shape and the type of information they convey Figure 1. Because the muscle spindle is located in parallel with the extrafusal fibers, it will stretch along with the muscle.
The muscle spindle signals muscle length and velocity to the CNS through two types of specialized sensory fibers that innervate the intrafusal fibers. These sensory fibers have stretch receptors that open and close as a function of the length of the intrafusal fiber. Because of their patterns of innervation onto the three types of intrafusal fibers, Group Ia and Group II afferents respond differently to different types of muscle movements.
Initially, both Group Ia and Group II fibers fire at a certain rate, encoding the current length of the muscle. During the stretch, the two types differ in their responses. The Group Ia afferent fires at a very high rate during the stretch, encoding the velocity of the muscle length; at the end of the stretch, its firing decreases, as the muscle is no longer changing length.
Note, however, that its firing rate is still higher than it was before the stretch, as it is now encoding the new length of the muscle. The Group II afferent increases its firing rate steadily as the muscle is stretched. Its firing rate does not depend on the rate of change of the muscle; rather, its firing rate depends only on the immediate length of the muscle. The Group Ia afferent responds at a highest rate when the muscle is actively stretching, but also signals the static length of the muscle because of its innervation of the static nuclear bag fiber and the nuclear chain fiber.
The Group II afferent signals only the static length of the muscle, increasing its firing rate linearly as a function of muscle length. Although intrafusal fibers do not contribute significantly to muscle contraction, they do have contractile elements at their ends that are innervated by motor neurons. The muscle starts at a certain length, encoded by the firing of a Ia afferent. When the muscle is stretched, the muscle spindle stretches and the Ia afferent fires more strongly.
When the muscle is released from the stretch and contracts, the muscle spindle becomes slack, causing the Ia afferent to fall silent. The muscle spindle is rendered insensitive to further stretches of muscle. To restore sensitivity, gamma motor neurons fire and cause the spindle to contract, thereby becoming taut and able to signal the muscle length again.
The hypotheses were validated, suggesting that intrafusal fibers have greater capacities for growth, regeneration, and repair than do adjacent extrafusal fibers. During maturation, extrafusal and intrafusal fibers show similar trends of decreasing SC frequencies and concentrations and increases in myonuclear domains. Thus, extrafusal and intrafusal fibers alike should exhibit reduced capacities for growth, regeneration, and repair during maturation.
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