Neurotrophin-3 ameliorates sensory-motor deficits in Er81-deficient mice.

Two factors, the ETS transcription factor ER81 and skeletal muscle-derived neurotrophin-3 (NT3), are essential for the formation of muscle spindles and the function of spindle afferent-motoneuron synapses in the spinal cord. Spindles either degenerate completely or are abnormal, and spindle afferents fail to project to spinal motoneurons in Er81 null mice; however, the interactions between ER81 and NT3 during the processes of afferent neuron and muscle spindle development are poorly understood. To examine if overexpression of NT3 in muscle rescues spindles and afferent-motoneuron connectivity in the absence of ER81, we generated myoNT3;Er81(-/-) double-mutant mice that selectively overexpress NT3 in muscle in the absence of ER81. Spindle reflex arcs in myoNT3;Er81(-/-) mutants differed greatly from Er81 null mice. Muscle spindle densities were greater and more afferents projected into the ventral spinal cord in myoNT3;Er81(-/-) mice. Spindles of myoNT3;Er81(-/-) muscles responded normally to repetitive muscle taps, and the monosynaptic inputs from Ia afferents to motoneurons, grossly reduced in Er81(-/-) mutants, were restored to wild-type levels in myoNT3;Er81(-/-) mice. Thus, an excess of muscle-derived NT3 reverses deficits in spindle numbers and afferent function induced by the absence of ER81. We conclude that muscle-derived NT3 can modulate spindle density and afferent-motoneuron connectivity independently of ER81.

ETS transcription factor ER81 is required for the Pacinian corpuscle development.

ER81, a member of the ETS family of transcription factors, is involved in processes of specification of neuronal identity, control of sensory-motor connectivity, and differentiation of muscle spindles. Spindles either degenerate or are abnormal in mutant mice lacking ER81. We examined whether ER81 is required for the development of another class of mechanoreceptors, the Pacinian corpuscle. ER81 was expressed by the inner core cells of the corpuscles, as reflected by expression of the lacZ reporter gene in Er81(+/lacZ) mutants, thereby suggesting a role for ER81 in the corpuscle development. No Pacinian corpuscles or their afferent nerve fibers were present in the crus of Er81 null mice at birth. Legs of mutant embryos examined at E16.5 were also devoid of the corpuscles, but not of their afferents. Thus, Pacinian corpuscles do not form, and their afferents do not survive, in the absence of ER81. A deficiency of dorsal root ganglia neurons expressing calretinin, a marker for neurons subserving Pacinian corpuscles, correlated with the absence of corpuscles and their afferents in Er81 null mice. These observations indicate a requirement for ER81 in the assembly of Pacinian corpuscles and the survival of the sensory neurons that innervate them.

Age-related changes in nigrostriatal dopaminergic function are accentuated in +/- brain-derived neurotrophic factor mice.

The effects of a deletion for the brain derived neurotrophic factor (BDNF) allele (+/- BDNF) upon age-related changes in nigrostriatal dopaminergic (NSDA) function were assessed. Behavioral (beam crossing and spontaneous activity) and neurochemical (potassium-stimulated dopamine release from superfused striatum) measures were compared among Young (4-5 month), Middle (11-13 month) and Aged (19-21 month) +/- BDNF and their wild type littermate control (+/+ BDNF) mice. No statistically significant differences were obtained between +/+ and +/- BDNF mice at the Young age sampling period for any of the behavioral or neurochemical measures. Behavioral and neurochemical responses indices of NSDA function begin to diverge between +/+ and +/- Middle age BDNF mice and maximal differences were observed at the Aged period. For both movement and stereotypy times, scores obtained from +/+ mice were significantly decreased compared with +/- BDNF mice at the Aged period and center time scores of +/+ mice were decreased at both the Middle and Aged periods compared with +/- BDNF mice. Neurochemically, potassium-stimulated DA release of +/+ mice was significantly greater than +/- BDNF mice with maximal differences obtained at the Aged period. These results demonstrate marked differences in age-related changes of NSDA function between +/+ and +/- BDNF mice and suggest that the deletion of one allele for BDNF may make these mice more susceptible to age-related declines in NSDA function.

Pacinian corpuscle development involves multiple Trk signaling pathways.

The development of crural Pacinian corpuscles was explored in neonatal mutant mice lacking nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3) or neurotrophin-4 (NT4), or their cognate Trk receptors. Deficits of the corpuscles and their afferents were greatest in NT3, less in BDNF, and least in NT4 null mice. Deletion of NGF or p75(NTR) genes had little or no impact. No Pacinian corpuscles were present in NT3;BDNF and NT3;NT4 double or NT3;BDNF;NT4 triple null mice. Deficits were larger in NT3 than TrkC mutants and were comparable to deficits observed in TrkB or TrkA mutants. Afferents of all corpuscles coexpressed TrkA and TrkB receptors, and some afferents coexpressed all three Trk receptors. Our results suggest that multiple neurotrophins, in particular NT3, regulate the density of crural Pacinian corpuscles, most likely by regulating the survival of sensory neurons. In addition, NT3/TrkB and/or NT3/TrkA signaling plays a greater role than NT3/TrkC signaling in afferents to developing Pacinian corpuscles.

Formation of supernumerary muscle spindles at the expense of Golgi tendon organs in ER81-deficient mice.

ER81, a member of the ETS family of transcription factors, is essential for the formation of connections between sensory and motor neurons in the spinal cord. Mice lacking Er81 genes exhibit reduced monosynaptic sensory-motoneuron connectivity in response to muscle nerve stimulation. Proximal muscle nerve stimulation elicits fewer monosynaptic potentials than stimulation of distal nerves in hindlimbs, a deficit that is paralleled by a paucity of muscle spindles in proximal muscles (Arber et al., 2000). We examined whether a presence of spindles innervated by afferents in distal muscles correlated with the increased preservation of monosynaptic sensory-motor potentials in distal muscle nerves. Not only were spindles and Ia afferents present, but also they were supernumerary in distal muscles such as the soleus, medial gastrocnemius, and extensor hallucis longus. Concomitantly, a deficiency of Golgi tendon organs (GTOs) and Ib afferents was observed in distal muscles, as if supernumerary spindles formed at the expense of tendon organs in the absence of Er81. Thus, ER81 may be involved in mechanisms that regulate acquisition of the Ia and Ib phenotypes by subsets of proprioceptive muscle afferents. Segmental differences in muscle spindle and GTO dependence on ER81 suggest that more than one ETS transcription factor may participate in the regulation of limb proprioceptive system assembly in the mouse.

Evaluation of nigrostriatal dopaminergic function in adult +/+ and +/- BDNF mutant mice.

Deletion of a single copy of the BDNF gene has been shown to affect the nigrostriatal dopaminergic system of young adult BDNF mice. In the present report we evaluated various indices of nigrostriatal dopaminergic function between 9-month-old wild-type (+/+) and heterozygous (+/-) BDNF mutant mice. Performance in a sensorimotor beam walking task was significantly decreased in +/- mice as indicated by increased times required to traverse both a wide (21 mm) and narrow (6 mm) beam. No differences in spontaneous locomotor behavior were observed between the +/+ and +/- mice. Amphetamine-stimulated (5 mg/kg) locomotor behavior was increased to a greater degree in the +/- mice, with the number of movements performed by these mice being significantly greater than their +/+ controls. Corpus striatal dopamine concentrations were significantly greater in the +/- BDNF mice. The absence of any significant differences for dopamine concentrations within the hypothalamus and olfactory bulb of these mice, as well as an absence of any difference in striatal norepinephrine concentrations, suggested a relative specificity of these effects to the corpus striatum. Both the +/- and +/+ mice showed similar reductions in striatal dopamine concentrations in response to a neurotoxic regimen of methamphetamine (20 mg/kg). Collectively these data show increased levels of striatal dopamine concentrations associated with altered behavioral responses involving the nigrostriatal dopaminergic system within the heterozygous BDNF mutant mice.

Inactivation of one copy of the mouse neurotrophin-3 gene induces cardiac sympathetic deficits.

Whether two copies of the neurotrophin-3 (NT3) gene are necessary for proper development of cardiac sympathetic innervation was investigated in mice carrying a targeted inactivation of the NT3 gene. Heterozygous (+/-) and null (-/-) mutant mice had fewer stellate ganglion neurons than did wild-type (+/+) mice at postnatal day 0 (P0 or birth), and this deficit was maintained between adult (P60) +/- and +/+ mice. The sympathetic innervation of the heart matured postnatally in +/+ and +/- mice. Tyrosine hydroxylase (TH)-positive axons were restricted largely to the epicardium at P0, were concentrated around large blood vessels in the myocardium at P21, and were present among cardiac myocytes at P60. Cardiac norepinephrine (NE) concentrations paralleled the growth of the sympathetic axons into the heart. NE concentrations were equivalent among +/+, +/-, and -/- mice at birth, but differences between +/- and +/+ mice increased with age. Adult +/- mice also exhibited lower resting heart rates and sympathetic tonus than +/+ mice. Thus deletion of one copy of the NT3 gene translates into anatomical, biochemical, and functional deficits in cardiac sympathetic innervation of postnatal mice, thereby indicating a gene-dosage effect for the NT3 gene.

Formation of a full complement of cranial proprioceptors requires multiple neurotrophins.

Inactivation of neurotrophin-3 (NT3) completely blocks the development of limb proprioceptive neurons and their end organs, the muscle spindles. We examined whether cranial proprioceptive neurons of the trigeminal mesencephalic nucleus (TMN) require NT3, brain-derived neurotrophic factor (BDNF) or neurotrophin-4 (NT4) for their development. Complements of TMN neurons and masticatory muscle spindles were decreased by 62% in NT3 null mutants, 33% in BDNF null mutants, and 10% in NT4 null mutant mice at birth. The extent of proprioceptive deficiencies differed among different masticatory muscles, particularly in NT3 null mice. Masticatory muscles of embryonic mice heterozygous for the NT3(lacZneo) or BDNF(lacZ) reporter genes expressed both NT3 and BDNF, consistent with target-derived neurotrophin support of TMN neurons. Although more than 90% of TMN neurons expressed TrkB as well as TrkC receptor proteins by immunocytochemistry in wild-type newborns, TrkC or TrkB null mice exhibited only partial proprioceptive deficiencies similar to those present in NT3 or BDNF;NT4 null mice. Thus, in terms of the survival outcome, two main subpopulations of TMN neurons may exist during embryogenesis, one dependent on TrkC/NT3 functioning and the other utilizing TrkB/BDNF signaling. The differential dependence of TMN neurons on neurotrophins may reflect differential accessibility of the neurons to limiting amounts of NT3, BDNF, or NT4 in target tissues, especially if the tissue distribution or levels of BDNF, NT3, and NT4 were dynamically regulated both spatially and temporally.

Why adult mammalian intrafusal and extrafusal fibers contain different myosin heavy-chain isoforms.

Multiple isoforms of the contractile protein myosin are present in mammalian skeletal muscles. The diversity of the heavy-chain subunits of myosin (MyHCs) in intrafusal fibers is thought to reflect a pathway of differentiation that is unique to muscle spindles. In fact, intrafusal MyHCs are developmental isoforms expressed by the prenatal precursors of both intrafusal and extrafusal fibers. In adult limbs, developmental MyHCs persist in intrafusal, but not extrafusal fibers principally due to the afferent neurons that arrest their maturational replacement by MyHCs associated with faster shortening velocities. The slow shortening velocities that are characteristic of developmental MyHCs might be adaptive for precise calibration of muscle spindles as sense organs.

Alterations in nigrostriatal dopaminergic function within BDNF mutant mice.

The influence of brain-derived neurotrophic factor (BDNF) upon the nigrostriatal dopaminergic system was evaluated in weanling and adult mice carrying a targeted inactivated BDNF gene. Regional specificity of this BDNF mutation was assessed by assaying catecholamine concentrations within the corpus striatum, hypothalamus, and olfactory bulbs. In weanling mice dopamine, but not norepinephrine, concentrations within the corpus striatum of homozygous mutant (-/-) mice were significantly reduced with levels being 54% that of the wild-type controls (+/+) and 49% that of the heterozygous mutant (+/-) mice. While no differences were obtained among the three genotypes for hypothalamic dopamine, norepinephrine concentrations of -/- mice were significantly lower, being 62% of +/+ mice and 49% of +/- mice. The dopamine concentrations of -/- mice within the olfactory bulb were significantly reduced (69%) compared to the +/-, but not +/+ mice. Olfactory bulb norepinephrine concentrations showed a statistically significant difference among each of the three conditions with minimal levels in -/- mice (62% of +/+ and 45% of +/-). In the adults, catecholamine concentrations were measured only in +/+ and +/- mice since -/- mice do not typically survive past 21 days. Dopamine, but not norepinephrine, concentrations within the corpus striatum were significantly increased (116%) in +/- compared to +/+ mice. No other statistically significant differences were obtained in catecholamine concentrations within the hypothalamus or olfactory bulb in these adult mice. These results show that homozygous BDNF mutations produce severe depletions within the nigrostriatal dopaminergic system and substantial reductions of norepinephrine within the hypothalamus and olfactory bulb. Interestingly, maximal catecholamine concentrations for all areas sampled at both ages were observed in the +/- mice. These latter findings may indicate some subtle changes in catecholamine functions resulting from a heterozygous BDNF mutation.

Development of fusimotor innervation correlates with group Ia afferents but is independent of neurotrophin-3.

Fusimotor neurons, group Ia afferents and muscle spindles are absent in mutant mice lacking the gene for neurotrophin-3 (NT3). To partition the effect of Ia afferent or spindle absence from that of NT3 deprivation on fusimotor neuron development, we examined the fusimotor system in a mutant mouse (NesPIXpNT3) that lacks Ia afferents and spindles, but has normal or elevated tissue levels of NT3 during embryogenesis. Fusimotor fibers were absent in lumbar ventral spinal roots, and limb muscles were devoid of Ia afferents and spindles in adult NesPIXpNT3 mice. In contrast, no deficiency in motoneuron numbers was observed in the trigeminal nucleus which contains cell bodies of motor axons innervating muscles of mastication. Spindles and Ia afferents were also present in the masticatory muscles. Thus, the development and/or survival of fusimotor neurons correlates with the presence of Ia afferents and/or spindles, and not with the amount of NT3 in the spinal cord or muscle.

Neurotrophin-3 and trkC in muscle are non-essential for the development of mouse muscle spindles.

Neurotrophin-3 (NT3) or TrkC null mutant mice were examined for the presence of muscle spindles. Muscles of mastication, but not limbs, contained spindles in newborn and adolescent mutants. The intramuscular distribution and morphological properties of spindles in mutant masticatory muscles were indistinguishable from those of wild-type spindles. Intrafusal fibers of NT3- or trkC-deficient spindles-expressed the slow-tonic isoform of myosin heavy chains, characteristic of wild-type spindles. Sensory nerve endings were observed in spindles of mutants by electron microscopy. Thus, NT3 or trkC, which is expressed in wild-type spindles, may serve functions other than those related to spindle assembly. Presumably, proprioceptive neurons innervating jaw muscles are dependent on factors other than NT3 for survival and maintenance.

Afferent-inherent regulation of myosin heavy chain isoforms in rat muscle spindles.

Whether afferents exert their morphogenetic influence on spindles through release of trophic factors at intrafusal fiber junctions or via participation in proprioceptive pathways which modulate the motor activity to muscles was investigated by comparing myosin heavy chain (MHC) expression in intrafusal fibers after ablation of afferents (deafferentation, or DA) to the extensor digitorum longus (EDL) of adult rats or after ablation of the corresponding central processes of afferents to the spinal cord (central-process ablation, or CPA). DA and CPA elicited an exaggerated pedal plantarflexion, and hypertrophy of the EDL concomitant with atrophy of the soleus in the affected hindlimb. Frequencies and patterns of expression of seven MHCs expressed by intrafusal fibers in CPA muscles were indistinguishable from normal rats. However, frequencies and patterns of expression of several MHCs were abnormal following DA. Thus factors transported anterogradely from afferents to intrafusal fibers may regulate MHC expression in intrafusal fibers.

Stability of myosin heavy chain isoforms in selectively denervated adult rat muscle spindles.

BACKGROUND: Rat intrafusal fibers consist of multiple isoforms of myosin heavy chains (MHCs) whose expression involves complex interactions among motor neurons, sensory neurons, and muscle cells during spindle development. Little is known about the roles of sensory and motor innervation in regulating and maintaining expression of MHC isoforms in adult rat muscle spindles. METHODS: MHC expression was investigated in deafferented or deefferented adult rat muscle spindles by reacting transverse sections of spindles with a panel of monoclonal antibodies specific for different MHC isoforms. RESULTS: Deefferentation or deafferentation did not alter the number of intrafusal fibers expressing most MHC isoforms. However, the numbers of fibers expressing two MHC isoforms were altered in deefferented muscle spindles. Nuclear bag1 fibers ceased to express alpha-cardiac MHC and upregulated embryonic MHC after ablation of motor innervation. Likewise, bag2 and chain fibers downregulated avian neonatal/fast MHC following deafferentation, but chain fibers upregulated type 2A MHC and became more extrafusal-like in their pattern of MHC expression. CONCLUSIONS: These data indicate that (1) perturbations in spindle sensory and motor nerve supplies produce less severe alterations in MHC expression in mature intrafusal fibers than do similar lesions in developing intrafusal fibers and (2) MHC expression in intrafusal fibers reflects a combination of inductive and suppressive effects of motor and sensory neurons.

Expression of alpha-cardiac myosin heavy chain in normal and denervated rat muscle spindles.

Whether any fibers in rat hindlimb muscles express alpha-cardiac myosin heavy chain (MHC) is uncertain. Expression of alpha-cardiac MHC mRNA and the polypeptide for which it codes were examined in control and denervated rat muscle spindles using in situ hybridization (ISH) and immunocytochemistry (ICC). Both nuclear bag2 and bag1 intrafusal fibers in the extensor digitorum longus (EDL) muscles expressed alpha-cardiac MHC and its precursor mRNA. Furthermore, denervation of the hindlimb down-regulated alpha-cardiac MHC mRNA expression in rat nuclear bag intrafusal fibers, even though they continued to display a strong affinity for anti-alpha-cardiac MHC monoclonal antibody. These data show that (1) intrafusal fibers express the alpha-cardiac MHC gene; (2) innervation regulates alpha-cardiac MHC gene expression at a pre-translational level; and (3) ISH is more sensitive than ICC to changes in alpha-cardiac MHC gene expression in adult rat muscle spindles.

An immunocytochemical marker for early type I muscle fibers in the developing rat hindlimb.

Muscle fibers develop sequentially from several generations of myotubes that express specific isoforms of myosin heavy chain (MHC). We observed that the chicken-derived monoclonal antibody (mAb) S46 binds to myotubes of the fetal rat hindlimb in a specific temporal and spatial pattern. To determine the type and fate of the S46-reactive myotubes, we immunoreacted sections of fetal, neonatal and postnatal hindlimb muscles to this antibody. The mAb S46 bound to a subpopulation of primary myotubes in the tibialis anterior, and to all primary and slow/fast secondary myotubes in the soleus muscle. The S46-reactive primary myotubes represented the oldest set of myotubes in the muscles. Reactivity to S46 was present from the earliest stages of muscle development, peaked in the late fetal period, and dissipated in the first postnatal week, suggesting that mAb S46 binds to a developmental form of slow myosin. The regional distribution of myotubes that bound S46 in fetal muscles was identical to the distribution of type I (slow-twitch) fibers in the adult, indicating that S46-reactive myotubes ultimately develop into type I extrafusal fibers. Thus, mAb S46 can be used as a marker for prospective type I extrafusal fibers in the rat hindlimb.

Origin of intrafusal fibers from a subset of primary myotubes in the rat.

S46, a monoclonal antibody (mAb) specific for the SM-1 and SM-2 isoforms of avian slow myosin heavy chains (MHC), was used to study the earliest stages of development of intrafusal fibers in muscle spindles of the rat hindlimb. Spindles formed only in the regions of fetal muscles that contained primary myotubes reactive to mAb S46, such as the axial region of the tibialis anterior muscle. The first intrafusal fiber to form, the nuclear bag2 fiber, originated from within the population of S46-reactive primary myotubes. Binding of mAb S46 by myotubes giving rise to the bag2 fibers preceded the appearance of encapsulated spindles in the muscles by electron microscopy. However, reactivity to S46 intensified in the myotubes transforming into bag2 fibers after the innervation of the fibers by afferents, and dissipated in myotubes differentiating into slow-twitch (type I) extrafusal fibers. Thus, afferents may enhance intrafusal expression of the MHC isoform reactive to mAb S46. The pattern of S46 binding to nuclear bag and chain intrafusal fibers in both developing and adult spindles was the same as that reported for the mAb ALD19, suggesting that both antibodies bind to the same MHC isoform. This isoform is probably a developmental form of slow myosin, because it was transiently expressed during the development of type I extrafusal fibers. The origin of bag2 intrafusal and type I extrafusal fibers from a bipotential subpopulation of primary myotubes reactive to mAb S46 correlates with the location of muscle spindles in the slow regions of muscles in adult rat hindlimbs.

Oxidative capacity of human muscle fiber types: effects of age and training status.

Morphometry and oxidative capacity of slow-twitch (type I) and fast-twitch (type IIa and IIb) muscle fibers obtained from vastus lateralis needle biopsies were compared between younger (21-30 yr) and older (51-62 yr) normal fit (maximal O2 uptake = 47.0 vs. 32.3 ml.kg-1.min-1) and endurance-trained (66.3 vs. 52.7 ml.kg-1.min-1) men (n = 6/group). The older groups had smaller type IIa (31%) and IIb (40%) fiber areas and fewer capillaries surrounding these fibers than did younger groups. The reduced type II fiber areas and capillary contacts associated with aging were also observed in the older trained men. However, the capillary supply per unit type II fiber area was not affected by age but was enhanced by training. Additionally, on the basis of quantitative histochemical analysis, succinate dehydrogenase activities of type IIa fibers in the older trained men [4.07 +/- 0.68 (SD) mmol.min-1.l-1] were similar to those observed in younger trained men (4.00 +/- 0.48 mmol.min-1.l-1) and twofold higher than in older normal fit men (2.01 +/- 0.65 mmol.min-1.l-1; age x fitness interaction, P < 0.05). Type I muscle fibers were unaffected by age but were larger and had more capillary contacts and higher succinate dehydrogenase activities in the trained groups. The findings of this study suggest that aging results in a decrease in type II fiber size and oxidative capacity in healthy men and that this latter effect can be prevented by endurance training. Conclusions regarding the effects of age and training status on muscle capillarization depend largely on how these data are expressed.

Sequences of intrafusal fiber formation are muscle-dependent in rat hindlimbs.

A rat muscle spindle typically contains four intrafusal fibers-one nuclear bag2, one nuclear bag1 and two nuclear chain fibers. We compared the sequence of formation of the three intrafusal fiber types among the tibialis anterior (TA), soleus (SOL) and medial gastrocnemius (MG) muscles using immunocytochemistry of spindle-specific myosin heavy chain isoforms. Spindles of the TA began to differentiate earlier and acquired the full complement of intrafusal fibers sooner than spindles of the SOL or MG muscles. At the onset of spindle assembly, the intrafusal myotubes expressed myosin heavy chains similar to those expressed by extrafusal myotubes. The first intrafusal myotube then differentiated into the bag2 fiber regardless of the muscle. However, the fate of the second-forming intrafusal myotube varied among the muscles studied. It usually differentiated into a chain fiber in the TA, into a bag1 fiber in the SOL, and into either a bag1 or a chain in the MG. The fate of the third-forming intrafusal myotube was reciprocal to that of the second; i.e. in those spindles in which the bag1 fiber was second to form, a chain was third, and vice versa. The fourth and last intrafusal myotube gave rise to a chain fiber. The inter- and intramuscular variability in the fate of intrafusal myotubes of the second and third generation argues against the existence of a program intrinsic to the myotubes that would mandate their differentiation along specific paths. Rather, an extrinsic regulatory factor, probably associated with the primary afferent neuron, may govern differentiation of pluripotential myotubes into particular types of intrafusal fiber. The fate of the intrafusal myotubes might then depend on the timing of the regulatory effect of afferents relative to the stage of development of the intrafusal bundle.