Neonatal Denervation Research Articles

Sex-specific role of myostatin signaling in neonatal muscle growth, denervation atrophy, and neuromuscular contractures.

Neonatal brachial plexus injury (NBPI) causes disabling and incurable muscle contractures that result from impaired longitudinal growth of denervated muscles. This deficit in muscle growth is driven by increased proteasome-mediated protein degradation, suggesting a dysregulation of muscle proteostasis. The myostatin (MSTN) pathway, a prominent muscle-specific regulator of proteostasis, is a putative signaling mechanism by which neonatal denervation could impair longitudinal muscle growth, and thus a potential target to prevent NBPI-induced contractures. Through a mouse model of NBPI, our present study revealed that pharmacologic inhibition of MSTN signaling induces hypertrophy, restores longitudinal growth, and prevents contractures in denervated muscles of female but not male mice, despite inducing hypertrophy of normally innervated muscles in both sexes. Additionally, the MSTN-dependent impairment of longitudinal muscle growth after NBPI in female mice is associated with perturbation of 20S proteasome activity, but not through alterations in canonical MSTN signaling pathways. These findings reveal a sex dimorphism in the regulation of neonatal longitudinal muscle growth and contractures, thereby providing insights into contracture pathophysiology, identifying a potential muscle-specific therapeutic target for contracture prevention, and underscoring the importance of sex as a biological variable in the pathophysiology of neuromuscular disorders.

Timing of proteasome inhibition as a pharmacologic strategy for prevention of muscle contractures in neonatal brachial plexus injury.

Neonatal brachial plexus injury (NBPI) causes disabling and incurable contractures, or limb stiffness, which result from proteasome‐mediated protein degradation impairing the longitudinal growth of neonatally denervated muscles. We recently showed in a mouse model that the 20S proteasome inhibitor, bortezomib, prevents contractures after NBPI. Given that contractures uniquely follow neonatal denervation, the current study tests the hypothesis that proteasome inhibition during a finite window of neonatal development can prevent long‐term contracture development. Following neonatal forelimb denervation in P5 mice, we first outlined the minimum period for proteasome inhibition to prevent contractures 4 weeks post‐NBPI by treating mice with saline or bortezomib for varying durations between P8 and P32. We then compared the ability of varying durations of longer‐term proteasome inhibition to prevent contractures at 8 and 12 weeks post‐NBPI. Our findings revealed that proteasome inhibition can be delayed 3‐4 days after denervation but is required throughout skeletal growth to prevent contractures long term. Furthermore, proteasome inhibition becomes less effective in preventing contractures beyond the neonatal period. These therapeutic effects are primarily associated with bortezomib‐induced attenuation of 20S proteasome β1 subunit activity. Our collective results, therefore, demonstrate that temporary neonatal proteasome inhibition is not a viable strategy for preventing contractures long term. Instead, neonatal denervation causes a permanent longitudinal growth deficiency that must be continuously ameliorated during skeletal growth. Additional mechanisms must be explored to minimize the necessary period of proteasome inhibition and reduce the risk of toxicity from long‐term treatment.

Timing of Proteasome Inhibition for Prevention of Muscle Contractures in Neonatal Brachial Plexus Injury

Background: Neonatal brachial plexus injury (NBPI) causes disabling and incurable contractures, or limb stiffness, which result from proteasome-mediated protein degradation impairing the longitudinal growth of neonatally denervated muscles. We recently showed in a mouse model that the 20S proteasome inhibitor, bortezomib, prevents contractures after NBPI. Given that contractures uniquely follow neonatal denervation, the current study tests the hypothesis that proteasome inhibition during a finite window of neonatal development can prevent long-term contracture development. Methods: We used a mouse model of NBPI in which neonatal forelimb denervation at P5 causes shoulder and elbow contractures 4 weeks post-denervation. We first outlined the minimum period for proteasome inhibition to prevent contractures 4 weeks post-NBPI by treating mice with saline or bortezomib for varying durations between P8 and P32. We then compared the ability of varying durations of longer-term proteasome inhibition to prevent contractures at 8 and 12 weeks post-NBPI. Elbow and shoulder contractures, elbow flexor muscle length, and proteasome catalytic subunit activity were assessed. Findings: Proteasome inhibition can be delayed 3-4 days after denervation but is required throughout skeletal growth to prevent contractures long-term. Proteasome inhibition becomes less effective in preventing contractures beyond the neonatal period. The therapeutic effect of bortezomib is associated with attenuation of 20S proteasome β1 subunit activity. Interpretation: Temporary neonatal proteasome inhibition does not prevent contractures long-term. Instead, neonatal denervation causes a permanent longitudinal growth deficiency that must be continuously ameliorated during skeletal growth. Additional mechanisms must be explored to reduce the risk of toxicity from long-term proteasome inhibition. Funding Statement: This work was supported by grants to RC from the National Institutes of Health (NIH) (R01HD098280-01) and Pediatric Orthopaedic Society of North America (POSNA), as well as funding from the Cincinnati Children’s Hospital Division of Orthopaedic Surgery and Junior Cooperative Society. Declaration of Interests: The authors have declared that no conflict of interest exists. Ethics Approval Statement: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All mice were handled according to approved institutional animal care and use committee (IACUC) protocols (#2017-0084) of the Cincinnati Children’s Hospital Medical Center. All surgeries were performed under isoflurane anesthesia, and every effort was made to minimize suffering.

“Popeye muscle” morphology in OBPI elbow flexion contracture

Background: The pathophysiology of elbow flexion contracture (EFC) in obstetrical brachial plexus injury (OBPI) is not established. In basic science models, neonatal denervation leads to impaired muscle growth. In clinical studies, diminished growth is correlated with extent of denervation, and improved with surgical repair. In EFC, the biceps are clinically short and round vs the contralateral size, termed the “Popeye muscle”. The objective of this study was to determine if the biceps morphology (muscle belly and tendon length) in arms with EFC secondary to OBPI is different vs the contralateral. Methods: This is a retrospective matched-cohort study. Patients with unilateral EFC (>20°) secondary to OBPI were identified (median = 6.6 years, range = 4.7–16.8). A blinded radiologist used computed tomography to measure length of the biceps short head muscle belly, and tendon bilaterally using standardised anatomical landmarks. Results: Twelve patients were analyzed. The biceps muscle belly in the injured arm was shorter in all patients vs contralateral, mean difference = 3.6 cm (80%), p < 0.001. The biceps tendon in the injured arm was longer in all patients vs contralateral, mean difference = 1.13 cm (127%), p < 0.001. The total biceps length in the injured arm was shorter in all patients vs contralateral, mean difference = 2.5 cm (89%), p < 0.001. Conclusions: This is the first human study confirming growth discrepancy of an elbow flexor in EFC. Distinct biceps morphology is demonstrated, with a significantly shorter muscle belly and overall length, but longer tendon vs normal. This is termed the “Popeye muscle” for its irregular morphology. Findings are consistent with impaired limb growth in denervation.

N-methyl-d-aspartate receptor subunit composition in the rat trigeminal principal nucleus remains constant during postnatal development and following neonatal denervation

N-methyl- d-aspartate receptors (NMDARs) play a major role in various forms of developmental and adult synaptic plasticity (Lopez de Armentia M, Sah P (2003) J Neurosci 23:6876–6883). Activity-dependent shifts in NR2 subunits of the NMDARs have been proposed to be the molecular basis of critical period plasticity. Several supporting examples have been reported; however it is not clear whether the relationship between NMDAR subunit changes and neural plasticity are correlative or causal, nor whether such a relationship is universal across all sensory pathways with developmental plasticity. In the present study, we used voltage-clamp recording techniques to investigate whether subunit composition of NMDARs changes during development and after neonatal denervation in the principal sensory nucleus (PrV) of the trigeminal nerve. Relative AMPA receptor contribution to synaptic transmission increased linearly by the second postnatal week in the normal PrV. Denervation by peripheral nerve damage did not alter this process. We took the weighted decay time constant (τ w) of NMDAR-mediated EPSCs as an index for NMDAR subunit composition. The τ w measurement and Western blot analysis revealed that NMDARs contained both NR2A and NR2B subunits. The NR2A/NR2B ratio did not change during postnatal development or after neonatal denervation. Thus, critical period plasticity-related pattern formation in the PrV does not depend on changes in subunit composition of NMDARs. The mechanism underlying developmental synaptic plasticity in the PrV differs from those in higher trigeminal centers and other brain structures.

The Role of Noradrenergic Nerves in the Development of the Lymphoproliferative Disease in Fas-Deficient, lpr/lpr Mice

Lpr/lpr mice develop a lymphoproliferative, autoimmune, lupus-like disease. These mice lack functional Fas (CD95) expression and are resistant to Fas ligand (CD178)-mediated apoptosis, a critical mechanism for the maintenance of peripheral tolerance. In this study, we show that noradrenaline (NA), the main sympathetic neurotransmitter, can induce apoptosis of lymphoid cells independently of functional Fas. Based on this finding, we used lpr/lpr mice as model to study the role of noradrenergic nerves in the expression of a lymphoproliferative disease. Early in ontogeny, the concentration of NA was significantly increased in the spleen of lpr/lpr mice, compared with normal littermates. However, splenic sympathetic innervation gradually declined as the disease progressed, and IgM blood levels and splenic NA concentration inversely correlated when the disease was overtly manifested. When the loss of noradrenergic fibers that occurred naturally during adult life in lpr/lpr mice was experimentally advanced by neonatal sympathectomy, the concentration of IgM and IgG2a in blood was markedly higher than that of control lpr/lpr mice, and the appearance of lymphadenopathy was accelerated. Furthermore, although neonatal denervation did not affect the life span of normal animals, it shortened significantly the survival time of lpr/lpr mice. These data show that, in addition to defects in the Fas pathway, an altered sympathetic innervation in lpr/lpr mice also contributes to the pathogenesis of the autoimmune disease, and strongly support the hypothesis that the sympathetic nervous system can modulate the expression of lymphoproliferative diseases.

Development of rodent macrovibrissae: Effects of neonatal whisker denervation and bilateral neonatal enucleation

In this paper we describe the effects of manipulating two kinds of sensory input in neonatal rats upon the development of the macrovibrissae—that movable subset of the rodent mystacial vibrissae. In an initial study of normal whisker development, data on whisker size were obtained from neonatal, perinatal, and adult rats. Data on whisker size were also obtained from rats sustaining either neonatal sensory or motor denervation of the whiskers and from both rats and mice bilaterally enucleated as neonates (BEN). In normally reared rats, most whiskers attain their final size over the first three postnatal weeks but development of rows 6 and 7 are not completed until after the first month. In normal animals we found a significant correlation both between body weight and whisker size and between the size of a whisker and the size of its corresponding cortical barrel. Rats sustaining neonatal denervation of the whiskers have shorter and thinner whiskers as adults than normally reared animals. In both rats and mice bilaterally enucleated as neonates a subset of the macrovibrissae are significantly larger than those of normal controls but no such effect is seen if the enucleation is carried out in adults. Moreover, BEN rats exposed to a novel stimulus environment whisk at a significantly higher frequency than normally reared animals. Mechanisms which might mediate these effects are discussed.

Anandamide elicits an acute release of nitric oxide through endothelial TRPV1 receptor activation in the rat arterial mesenteric bed

In the isolated rat mesenteric bed, the 1 min perfusion with 100 nm anandamide, a concentration that did not evoke vasorelaxation, elicited an acute release of 165.1 +/- 9.2 pmol nitric oxide (NO) that was paralleled by a 2-fold increase in cGMP tissue levels. The rise in NO released was mimicked by either (R)-(+)-methanandamide or the vanilloid receptor agonists resiniferatoxin and (E)-capsaicin but not by its inactive cis-isomer (Z)-capsaicin. The NO release elicited by either anandamide or capsaicin was reduced by the TRPV1 receptor antagonists 5'-iodoresiniferatoxin, SB 366791 and capsazepine as well as by the cannabinoid CB(1) receptor antagonists SR 141716A or AM251. The outflow of NO elicited by anandamide and capsaicin was also reduced by endothelium removal or NO synthase inhibition, suggesting the specific participation of endothelial TRPV1 receptors, rather than the novel endothelial TRPV4 receptors. Consistently, RT-PCR showed the expression of the mRNA coding for the rat TRPV1 receptor in the endothelial cell layer, in addition to its expression in sensory nerves. The participation of sensory nerves on the release of NO was precluded on the basis that neonatal denervation of the myenteric plexus sensory nerves did not modify the pattern of NO release induced by anandamide and capsaicin. We propose that low concentrations of anandamide, devoid of vasorelaxing effects, elicit an acute release of NO mediated predominantly by the activation of endothelial TRPV1 receptors whose physiological significance remains elusive.

Reinnervation of the rat levator ani muscle after neonatal denervation

After axonal injury on postnatal day 14 (P14), but not P21, motoneurons in the spinal nucleus of the bulbocavernosus (SNB) do not display their normal response to circulating testosterone levels. This could result from a permanent disruption of communication between motoneurons and their testosterone-sensitive target muscles. We assessed the extent of reinnervation of one of these target muscles, the levator ani (LA) muscle, 5 months after the pudendal nerve was cut either on P14 or P21. The number of motoneurons innervating the LA in control and nerve cut animals was determined using retrograde labeling procedures. Functional recovery of the LA muscle was determined via the testing of its in situ contractile properties. Compared to control muscles, reinnervated LA muscles were smaller, had fewer muscle fibers, generated a lower maximum tetanic tension, and were more fatigable. In spite of the fact that fewer motoneurons reinnervated the LA muscle after nerve cut on P14 than on P21, there were no differences in the weight or contractile properties of the LA muscle between these two groups. These data suggest that motoneurons that survived injury on P14 innervated more muscle fibers than normal and exhibited a similar ability to functionally reinnervate the target muscle as those motoneurons that survived injury on P21.

Brain sites of movement disorder: genetic and environmental agents in neurodevelopmental perturbations.

In assessing and assimilating the neurodevelopmental basis of the so-called movement disorders it is probably useful to establish certain concepts that will modulate both the variation and selection of affliction, mechanisms-processes and diversity of disease states. Both genetic, developmental and degenerative aberrations are to be encompassed within such an approach, as well as all deviations from the necessary components of behaviour that are generally understood to incorporate "normal" functioning. In the present treatise, both conditions of hyperactivity/hypoactivity, akinesia and bradykinesia together with a constellation of other symptoms and syndromes are considered in conjunction with the neuropharmacological and brain morphological alterations that may or may not accompany them, e.g. following neonatal denervation. As a case in point, the neuroanatomical and neurochemical points of interaction in Attention Deficit and Hyperactivity disorder (ADHD) are examined with reference to both the perinatal metallic and organic environment and genetic backgrounds. The role of apoptosis, as opposed to necrosis, in cell death during brain development necessitates careful considerations of the current explosion of evidence for brain nerve growth factors, neurotrophins and cytokines, and the processes regulating their appearance, release and fate. Some of these processes may possess putative inherited characteristics, like alpha-synuclein, others may to greater or lesser extents be endogenous or semi-endogenous (in food), like the tetrahydroisoquinolines, others exogenous until inhaled or injested through environmental accident, like heavy metals, e.g. mercury. Another central concept of neurodevelopment is cellular plasticity, thereby underlining the essential involvement of glutamate systems and N-methyl-D-aspartate receptor configurations. Finally, an essential assimilation of brain development in disease must delineate the relative merits of inherited as opposed to environmental risks not only for the commonly-regarded movement disorders, like Parkinson's disease, Huntington's disease and epilepsy, but also for afflictions bearing strong elements of psychosocial tragedy, like ADHD, autism and Savantism.

Deprivation and denervation differentially affect zinc-containing circuitries in the barrel cortex of mice

In the neocortex, a population of glutamatergic synapses contains chelatable zinc that is released upon depolarization. The present study compares the effect of chronic tactile deprivation and vibrissectomy performed at different postnatal ages on the synaptic zinc distribution in the mouse barrel cortex. We found that a chronic unilateral tactile deprivation resulted in an increase of synaptic zinc in deprived barrels. Distribution and intensity of zinc staining in non-deprived barrels resembled the control situation. The increase of zinc staining was observed if chronic deprivation started in early postnatal life or in adolescent mice but not in 70-day-old animals. This suggests that a critical period exists for plasticity of zinc containing terminals in the barrel cortex. The alteration of zinc staining was localized to not only the thalamorecipient layers IV but also layer II/III, and upper layer V. Neonatal denervation of selected vibrissal rows resulted in rearrangement of synaptic zinc distribution following cytoarchitectonic alterations in the barrel field. However, no changes in the intensity of zinc staining were observed. Vibrissectomy performed after the critical period for barrel formation did not affect either the distribution or intensity of zinc staining. It appears that the integrity of vibrissa-barrel pathway is necessary to induce activity-dependent alterations in synaptic zinc.

Involvement of NGF in the induction of increased noradrenergic innervation of the ureter in neonatally capsaicin-treated rats

Neonatal denervation of primary afferents with capsaicin leads to increased sympathetic innervation of the rat ureter. In the present study the development and the immunohistochemical characterization of this sympathetic hyperinnervation as well as the specific involvement of nerve growth factor (NGF) was investigated. Noradrenaline levels were found elevated in neonatally capsaicin-treated rats by 2 weeks of age and remained at that high level into adulthood. Injections of an anti-NGF antiserum during postnatal days (PN) PN 8–14, PN 13–19 or during PN 17–23 counteracted the capsaicin effect and reduced noradrenaline towards control levels. Immunohistochemical localization of tyrosine hydroxylase (TH), a marker for sympathetic nerve fibres, revealed that the capsaicin-induced hyperinnervation was mainly represented by fibres in deeper muscle layers and to a smaller extent by fibres in the submucosa. In control animals and in rats treated with capsaicin and anti-NGF antiserum fibres were mainly distributed in the adventitia and in the outer part of the smooth muscle layer. These results show that NGF is responsible for the development of an increased noradrenergic innervation in the rat ureter after neonatal capsaicin treatment.

Functional and Molecular Differentiation of the Dopamine System Induced by Neonatal Denervation

JOYCE, J. N., P. A. FROHNA AND B. S. NEAL-BELIVEAU. Functional and molecular differentiation of the dopamine system induced by neonatal denervation. NEUROSCI BIOBEHAV REV 20(3)453–486, 1996.—The administration of the neurotoxin 6-hydroxydopamine (6-OHDA) to damage the mesostriatal dopamine (DA) system in the neonate results in different neurochemical and behavioral consequences as compared to lesions made in adulthood. There have been few direct data to support the conclusion that the behavioral changes following neonatal 6-OHDA lesions reflect plasticity of the DA system. It is our hypothesis that the plasticity of the developing DA system is fundamentally different from that of the adult. Responses to 6-OHDA lesions can only be understood within the context of the status of the mesostriatal DA system at the time of the lesion. There are stages of development in the early postnatal period when certain components of the mesostriatal DA system are differentially sensitive to 6-OHDA lesions. These “windows” of vulnerability can be predicted from an analysis of the developmental expression of DA receptors and the maturation of the subpopulation of the mesostriatal DA system that innervates them. We review the differences in the behavioral plasticity of the adult and neonate sustaining 6-OHDA lesions to the mesostriatal DA system, the mechanisms responsible for the behavioral plasticity in the adult, and our conceptualization of which mechanisms are affected in the neonate. Copyright © Elsevier Science Ltd.

Schwann cell apoptosis at developing neuromuscular junctions is regulated by glial growth factor.

Denervated adult mammalian muscle fibres are reinnervated by regenerating axons and, in the case of partially denervated muscles, by sprouts extended from remaining, intact axons. Recent experiments suggest that Schwann cells (SCs) regulate these events, inducing and guiding axonal outgrowth through the processes they extend. In contrast to adults, reinnervation o denervated neonatal muscles is deficient and axonal sprouting is absent. In light of the proposed roles for SCs in these processes, we examined whether SCs in neonatal muscles exhibit altered responses to denervation. We report here that neonatal denervation leads to the rapid, apoptotic death of SCs at rat neuro-muscular junctions. Injection of glial growth factor, a member of the neuregulin family of trophic factors present in developing sensory and motor neurons, prevents this apoptosis in vivo. These results provide further evidence for the importance of SCs in regulating nerve growth and suggest that axon-Schwann cell trophic interactions play a role in the normal development of the neuromuscular system.

Satellite cells and myonuclei in long-term denervated rat muscles.

The percentage of satellite cells rapidly decreases in aneurally regenerating soleus muscles of rat. Also denervation of intact muscles causes fiber loss and regeneration, but the fate of satellite cells is unknown; myonuclei have been suggested to undergo changes resembling those in apoptotic cells. Rat soleus and extensor digitorum longus (EDL) muscles were denervated at birth or at age 5 weeks and investigated after periods of up to 38 weeks. At least 400 myonuclei in each muscle were assessed by electron microscopy, and satellite cell nuclei were counted. In situ nick translation and tailing were performed after 30 weeks denervation in order to demonstrate DNA breaks associated with apoptosis. Myotubes indicating regeneration were prominent in the adult denervated soleus and deep layers of EDL muscles after 7 weeks and in the superficial parts of EDL muscle after 16 weeks. The percentage of satellite cell nuclei slowly decreased to less than one fifth of normal after 20-30 weeks. Almost all satellite cells had vanished 10 weeks after neonatal denervation. Degenerating myonuclei in adult, but not in neonatally denervated muscles, remotely resembled apoptotic nuclei of lymphocytes, but no evidence of DNA breaks was found. Denervation of rat skeletal muscles causes, in addition to fiber atrophy, loss of fibers with subsequent regeneration. Proliferation of satellite cells under aneural conditions may lead to exhaustion of the satellite cell pool. This process is more rapid in growing than in adult muscles. Myonuclei in denervated muscles do not show DNA breaks which can be demonstrated by in situ nick translation.

Neural input and the development of adrenergic intracellular signaling: neonatal denervation evokes neither receptor upregulation nor persistent supersensitivity of adenylate cyclase

In the adult, denervation of adrenergic target tissues leads to compensatory upregulation of receptor sites and to supersensitive responses. When 6-hydroxydopamine (6-OHDA) was given to neonatal rats, cardiac β-receptors failed to show significant upregulation throughout the first five postnatal weeks and α 1-receptors were unchanged except at 35 days of age, despite 70–95% depletion of norepinephrine. The failure to upregulate could not be attributed to the high background level of receptor expression commensurate with ontogenetic increases in receptor numbers, since the same deficiency was seen in the liver, a tissue in which β-receptors decline with development; liver at-receptors also failed to upregulate after neonatal denervation. Examination of the linkage of β-receptors to adenylate cyclase indicated major differences from mature regulatory mechanisms, as denervation supersensitivity was completely absent (liver) or emerged only transiently several weeks after 6-OHDA treatment (heart). In the heart, there was evidence for a defect in the G-protein-dependent component of the receptor/cyclase linkage that could contribute to the delayed appearance of supersensitivity. Because the fundamental patterns of receptor ontogeny and of adenylate cyclase responsiveness are still present after neonatal denervation, it is unlikely that neural input provides the major impetus for basal development. However, adult-type regulation of receptors and responses did not emerge even after a prolonged period; thus, neural input during a critical developmental stage may be required for the cell to learn how to adjust receptor expression and the receptor/cyclase link in response to stimulation.

Regulation of c-ski transgene expression in developing and mature mice

The control of c-ski transgene expression and muscle hypertrophy have been investigated in transgenic mice. In adult animals, the level of transgene expression is linked to the specialized phenotype of individual muscles, high levels occur in fast muscles and significantly lower levels in muscles with high metabolic activity (diaphragm, soleus). These findings have led us to propose that a threshold must be passed before ski-induced growth can occur. We now show that within fast muscles, induced hypertrophy uniquely involves IIb fibers. This pattern of expression is under development control; levels of c-ski mRNA are low in all muscles at birth. In the diaphragm, there is a sevenfold increase in c-ski message levels between 5 d and maturity. By contrast, in fast extensor digitorum longus and anterior tibial muscles, there is a 24-fold increase in levels between 5 and 12 d postpartum. Muscle hypertrophy and antibody staining for c-ski protein in myofiber nuclei emerge concurrently. This pattern of c-ski expression parallels the appearance of IIb myosin heavy chain transcripts (Wydert et al., 1987) and differentiation of IIb fibers, suggesting that amplification of c-ski mRNA levels is linked to the development of IIb fiber specialization. Manipulations that are known to perturb IIb fiber development, neonatal denervation, and neonatally induced hypothyroidism inhibit high levels of c-ski expression and hypertrophy. In the adult fast EDL, denervation leads to rapid atrophy of IIb fibers and a significant decline in levels of c-ski mRNA. The results suggest that the environment of differentiated IIb fibers permits the expression of high levels of c-ski mRNA and this, in turn, induces hypertrophy.

Morphology of A beta hair follicle afferent collaterals in dorsal horn of cats with neonatal chronic denervation of digits.

1. The effect of neonatal denervation of two adjacent hindpaw digits (digits 3 and 4) in the cat on the morphology of spinal collaterals of identified A beta hair follicle afferents (HFAs) with receptive fields (RFs) on the neighbouring digits has been investigated using intra-axonal injection of horseradish peroxidase. 2. The morphology of collaterals of HFAs innervating intact digits (digit 2 or digit 5) was examined within the neonatally and chronically deprived region of dorsal horn by selectively injecting the afferents directly over this region. 3. Of a total of nine injected axons, seven had RFs on the distal part of digit 2, whereas two had RFs on the distal part of digit 5. With the exception of one of the digit 5 afferents, all injected afferents gave rise to stained collaterals throughout the chronically deprived digit representation. 4. Many collaterals within the chronically deprived region of dorsal horn gave rise to arborizations having relatively large numbers of boutons when compared with previously described somatotopically inappropriate (SIA) collaterals in the normal adult cat. 5. It is suggested that neonatal denervation of digits in the cat may lead to collateral sprouting and bouton formation by SIA collaterals of A beta HFAs innervating adjacent intact digits. This morphological plasticity could explain the previously reported functional reorganization of the RFs of spinocervical tract neurons that occurs in these neonatally deafferented animals.

Transient expression of a slow-tonic MHC isoform by extrafusal fibers in the developing rat.

ALD 19, a monoclonal antibody that recognizes the slow-tonic myosin heavy chain (MHC) isoform, has been used extensively as a marker for nuclear bag intrafusal fibers of muscle spindles in developing and adult rats. Extrafusal fibers of adult rat hindlimb muscles do not express slow-tonic MHC. However, while using ALD 19 to trace the fate of intrafusal fibers following neonatal denervation, we noted that some extrafusal fibers of neonates also bound this antibody. The immunolabeled extrafusal fibers were a subset of slow fibers located in the deep axial regions of crural muscles. The same fiber subset transiently displayed a weak affinity for ALD 19 during the first postnatal week in normal muscles. Denervation at birth increased the intensity of ALD 19 immunolabelling by these extrafusal fibers and extended the duration of the slow-tonic immunoreactivity into the 2nd postnatal week, after which expression diminished or ceased. Demonstration that some developing extrafusal fibers have a nerve-independent capacity for transiently expressing slow-tonic MHC, an MHC previously though to be expressed only by intrafusal fibers, raises the possibility that both types of fiber originate from a subset of bipotential slow primary myotubes in rat hindlimbs.

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.