Jon M. Walro

Superfluousness of motor innervation for the formation of muscle spindles in neonatal rats.

SummaryMuscle spindles form de novo in reinnervated muscles of neonatal rats treated with nerve growth factor. Whether the spindles can also form in muscle reinnervated only by afferents was investigated by removing the lumbosacral segment of the spinal cord immediately after crushing the nerve to the medial gastrocnemius muscle at birth, and administering nerve growth factor for 10 days afterwards. As predicted, the medial gastrocnemius muscles were reinnervated by afferents, but not efferents. No motor endplates were visible on any muscle fibers, and extrafusal fibers were atrophied. The reinnervated muscles contained spindle-like encapsulations of one to four fibers at 5, 7, 9 and 30 days after the nerve crush. The number of spindles as well as encapsulated fibers exceeded that of normal medial gastrocnemius muscles. The encapsulated fibers resembled typical intrafusal fibers. They had normal sensory-muscle contacts, but no motor endings. The fibers displayed equatorial clusters of myonuclei and expressed the spindle-specific slow-tonic myosin heavy chain isoform at postnatal day 30. Thus, efferents are not essential for the formation and differentiation of muscle spindles in reinnervated muscles of neonatal rats.

Postnatal expression of myosin heavy chains in muscle spindles of the rat

SummaryThe immunocytochemical expression of two myosin isoforms in intrafusal muscle fibers was examined in soleus muscles of neonatal (zero to six days postpartum) and adult rats. Monoclonal antibodies specific for myosin heavy chains of the slow-tonic anterior latissimus dorsi (ALD58) and fast-twitch pectoralis (MF30) muscles of the chicken were used. In adults ALD58 bound to the intracapsular regions of bag1 and bag2 fibers and MF30 bound to the intracapsular regions of bag2 and chain fibers. The extracapsular regions of intrafusal fibers and all extrafusal fibers did not react to ALD58 or MF30. Bag1 and bag2 fibers of neonatal rats expressed immature myosin patterns but chain fibers did not. The adult pattern of immunoreactivity of intrafusal fibers developed by the fourth postnatal day, when the patterns of motor but not sensory innervation in the spindle are still immature. Data suggest that the expression and maintenance of the specific anti-myosin immunoreactivity of intrafusal fibers during postnatal development of rat spindles is dependent upon sensory but not motor innervation. Moreover, afferents might regulate the gene expression responsible for synthesis of myosins isoforms specific to intrafusal fibers only in those myonuclei located within the capsule, but not in the myonuclei in extracapsular regions of intrafusal fibers.

Postnatal maturation of spindles in deafferented rat soleus muscles

SummaryWhether the motor innervation can direct the morphological and histochemical differentiation of developing muscle spindles in the absence of sensory innervation was investigated by deafferentation of the soleus muscle in immature rats. Dorsal root ganglia containing the cell bodies of afferents from the soleus muscle were removed surgically at a stage of postnatal development when spindles already contain the full complement of intrafusal fibers innervated by both afferents and efferents, but when the fibers are histochemically and structurally immature. Experimental soleus muscles were excised one year after deafferentation and sectioned frozen at a thickness of 8 μm. Sections were stained for enzymes indicative of types of muscle fibers and sites of neuromuscular junctions, and were examined by light microscopy. Spindles of muscles that matured in the absence of sensory innervation were abnormal. They lacked the periaxial fluid space and contained fewer intrafusal fibers than did normal spindles. The morphological and histochemical profiles of the encapsulated fibers present in the deafferented spindles more closely resembled extrafusal rather than intrafusal muscle fibers. These observations suggest that deafferentation of the immature spindles induces disintegration of some intrafusal fibers and alters maturation of others. Moreover, motor axons terminated less frequently along muscle fibers in deafferented spindles than on intrafusal fibers of normal spindles. Thus, maintenance of a full complement of intrafusal fibers in the developing spindle, emergence of histochemical profiles typical of normal intrafusal fibers, and development of adult pattern of fusimotor innervation require intact sensory innervation.

Differential effects of neonatal denervation on intrafusal muscle fibers in the rat

The response of developing muscle spindles to denervation was studied by sectioning the nerve to the medial gastrocnemius muscle of rats at birth. The denervated spindles were examined daily throughout the first postnatal week for changes in ultrastructure and expression of several isoforms of myosin heavy chain (MHC). Each of the three different types of intrafusal muscle fiber exhibited a different response to denervation. Within 5 days after the nerve section nuclear bag2 fibers degenerated completely; nuclear bag1 fibers persisted, but ceased to express the ‘spindle-specific’ slow-tonic MHC isoform and thereby could not be differentiated from extrafusal fibers; nuclear chain fibers did not form. The capsules of spindles disassembled, hence spindles or their remnants could no longer be identified 1 week after denervation. Neonatal deefferentation has little effect on these features of developing spindles, so removal of afferent innervation is presumably the factor that induces the loss of spindles in denervated muscles. Degeneration of the bag2 fiber, but not bag1 or extrafusal fibers, reflects a greater dependence of the bag2 fiber than the bag1 fiber on afferent innervation for maintenance of its structural integrity. This difference in response of the two types of immature bag fiber to denervation might reflect an origin of the bag2 fibers from a lineage of myogenic cells distinct from that giving rise to bag1 or extrafusal fibers, or a difference in the length of contact with afferents between the two types of bag fiber prior to nerve section.

Motor and sensory innervation of muscle spindles in the neonatal rat.

SummaryNeural and muscular elements of three muscle spindles from the soleus muscles of 4-day-old rats were reconstructed by electron microscopy of skip-serial transverse ultrathin sections. Each spindle contained four encapsulated intrafusal fibers, including a minimum of one bag1, one bag2 and one chain fiber. The fibers were innervated by unmyelinated motor and sensory axons. The primary and secondary afferents approached the spindles as single axons and terminated on the central region of the intrafusal fibers. Single profiles of terminal axons occupied the sites of sensory neuromuscular junctions, similar to adult sensory endings. No morphological features suggested retraction of afferents from 4-day postnatal spindles. Motor axons approached spindles tightly packed in bundles of 5–20 axons and terminated in the juxtaequatorial and polar regions of both bag and chain fibers. Multiple profiles of terminal axons were visible for each intrafusal motor ending. More motor axons innervated 4-day postnatal spindles and a greater number of axon terminals were visible in immature intrafusal motor endings than in adult spindles. The data suggest that postnatal maturation of motor innervation to intrafusal fibers involves the elimination of supernumerary motor nerve inputs. Synapse elimination in the development of the fusimotor system might represent a mechanism whereby individual γ axons adjust the number of spindles they innervate.

Myosin heavy chain expression in developing rat intrafusal muscle fibers

The immunocytochemical expression of several isoforms of myosin heavy chains (MHC) was determined in developing intrafusal and extrafusal fibers of the soleus muscle of prenatal and postnatal rats. At the onset of spindle assembly, both bag2 intrafusal myotubes and primary extrafusal myotubes bound a slow-twitch MHC antibody, whereas the bag1 and chain myotubes expressed a fast-twitch MHC isoform identical to that expressed by secondary extrafusal myotubes. Subsequently, developing intrafusal fibers began to express unique myosin isoforms, and ceased to express some of the myosin isoforms present initially. The initial similarity in MHC composition of intrafusal and extrafusal fibers suggests that these two kinds of mammalian muscle cell originate from a common pool of bipotential myotubes. Differences in MHC expression by intrafusal and extrafusal fibers in adult muscles might result from the effect of sensory neurons on the developing intrafusal myotubes.

Axotomy induces fusimotor-free muscle spindles in neonatal rats

Crushing the nerve to the medial gastrocnemius (MG) muscle at birth and administering nerve growth factor to rats afterwards results in a reinnervated muscle with supernumerary muscle spindles, some of which must have formed de novo. Structure and innervation of spindles in the reinnervated MG muscles were studied in serial 1 micron transverse sections. Two types of spindle-like encapsulations were observed. The prevalent type consisted of one to three small diameter intrafusal fibers with features of nuclear chain fibers or infrequently a nuclear bag fiber. The second type of encapsulation consisted of the small-diameter fibers located in a compartment which abutted a compartment containing a large diameter extrafusal fiber. All intrafusal fibers in spindles of the experimental muscles were innervated by afferents, but most of them (85%) were devoid of efferent innervation. Thus, immature fusimotor neurons may be more susceptible than spindle afferents to cell death after axotomy at birth.

The effect of neonatal deafferentation or deefferentation on the immunocytochemistry of muscle spindles in the rat

Immature muscle spindles were either deafferented or deefferented by selectively severing the sensory or motor nerve supply to the soleus muscle in neonatal rats. Experimental spindles were examined two months after the surgery using monoclonal antibodies specific for myosin heavy chains of slow-tonic and fast-twitch chicken muscles. The deefferented spindles exhibited a pattern of antibody binding that closely resembled that of normal adult intrafusal fibers, whereas deafferented intrafusal fibers were unreactive with the two antibodies. These observations suggest that sensory innervation is responsible for the expression of myosins in developing intrafusal muscle fibers of rat.

Formation of muscle spindles in the absence of motor innervation

Whether muscle spindles can form in muscles innervated only by afferents was investigated by removing the lumbosacral segment of the spinal cord immediately after crushing the nerve to the medial gastrocnemius (MG) muscle in newborn rats, and administering nerve growth factor for 10 days afterwards. The nerve-crushed MG muscles reinnervated by afferents in the absence of motor innervation were examined at postnatal (P) days 7, 9 and 30 for the presence of spindles by light and electron microscope. Reinnervated MG muscles contained spindle-like encapsulations of 1-4 fibers at 7, 9 and 30 days after the nerve crush. The number of spindles exceeded that of normal MG muscles, suggestive of de novo formation of spindles. All nerve-muscle contacts in the spindles had features of sensory endings, and intrafusal fibers expressed the spindle-specific slow-tonic myosin heavy chain (MHC) isoform at P30. No motor endplates were visible on any muscle fibers and extrafusal fibers were atrophied, as would be predicted in the absence of motor innervation. Thus, efferents are not essential for the formation and differentiation of muscle spindles in reinnervated muscles of neonatal rats.

MUSCLE SPINDLES FORM IN PARALYZED BUT NOT IN ANEURAL HINDLIMBS OF FETAL RATS

The necessity of innervation and/or neural activity for the formation of muscle spindles was investigated by treating fetal rats with neurotoxins on embryonic day 16 or 17 (E16-17), one or two days prior to the onset of spindle assembly. The neurotoxin-treated soleus muscles were examined on E21 for the presence of spindles and immunocytochemical expression of the slow-tonic myosin heavy-chain (MHC) isoform, which is characteristic of intrafusal fibers. Irreversible destruction of sensory and motor nerves by beta-bungarotoxin prevented the formation of spindles and expression of the slow-tonic MHC. Abolishment of nerve and muscle activity by tetrodotoxin did not block the spindle assembly or expression of the slow-tonic MHC. Thus, the formation and differentiation of spindles is dependent on innervation, but is independent of activity in nerve fibers or muscle cells.