Program :    Biomedical Research and Countermeasures	Ground Research
Element :    Physiology
Motoneuron Influences on Muscle Atrophy in Simulated Microgravity
Principal Investigator:
Dennis R. Mosier, M.D., Ph.D. NB302 Baylor College of Medicine One Baylor Plaza Houston, TX 77030	Phone: (713) 798-5971 Email: dmosier@bcm.tmc.edu Fax: (713) 798-3853 Congressional District: TX-25
Co-Investigator(s):
Appel, Stanley H. Booth, Frank W. Schwartz, Robert J. Baylor College of Medicine University of Missouri (Columbia) Baylor College of Medicine
Monitoring Center: NSBRI	Solicitation: NSBRI
Initial Funding Date:	Expiration:
Students Funded Under Research: 0	Post-Doctoral Associates: 2
Task Description:
Many alterations in motor unit structure and function occur with exposure to microgravity during spaceflight, and could impair motor performance. While much work is ongoing to ascertain the nature of biochemical, structural, and physiological changes occurring in muscle fibers, little attention has been paid to the changes reported in motoneuron terminals at the skeletal neuromuscular junction, and in motoneuron cell bodies, during exposure to microgravity. It is unlikely that these changes, whether they occur independently or secondary to changes in the innervated muscle fibers, are without consequences for the regulation of motor unit function. Accordingly, the central hypothesis of this study is that alterations in motoneuron structure and function occur during the process of microgravity-induced muscle atrophy, and that these alterations significantly influence muscle dysfunction, adaptation, and recovery from atrophy induced by microgravity. These changes may be manifested as early structural and functional alterations in the distal motoneuron terminal, in addition to alterations in motoneuron activity produced by changes in stretch reflexes and supraspinal pathways. Initiation of alterations in motoneuron terminals may be influenced by retrograde signals from muscle which induce, as an early event, changes in intracellular calcium and transmitter release. To begin to address these hypotheses, a combination of electrophysiologic assays of transmitter release at neuro-muscular junctions, coupled with electron microscopic assays of junctional remodelling, synaptic vesicles, and intraterminal calcium, is being used to define quantitatively the nature, extent, and possible significance of changes in motoneuron terminals occurring in a mouse model of unloading-induced muscle atrophy. During the course of this work, a technique for S-SFEMG (stimulated single-fiber electromyography) was adapted and validated for mice, allowing in vivo measurements of neuromuscular transmission. Data from this work suggest that: (1) unloading of skeletal muscle is associated with altered transmitter release at neuromuscular junctions, ultrastructural abnormalities, and a reduced safety factor suggesting insecure neuromuscular transmission; (2) the extent and nature of these junctional alterations vary among individual hindlimb muscles, possibly relating to differences in muscle loading and/or fiber type composition; (3) the extent of junctional alterations varies with duration of hindlimb unloading; (4) unloading of skeletal muscle may be associated with increased calcium in the motoneuron terminal, which may act as a signal inducing the observed junctional alterations; and (5) altered neuromuscular junctions in unloaded muscle retain the insensitivity to acute muscle stretch typical of normal mammalian junctions. Based on our observations, we hypothesize that junctional remodeling associated with muscle atrophy may vary over time, and may, especially in combination with other physiological stresses encountered during spaceflight (e.g., hypothermia, medication effects), pose a risk of junctional transmission failure. In the mouse hindlimb unloading model, we were unable to reproduce the full range of ultrastructural changes reported in studies of space-flown animals, and therefore suggest that additional factors (especially reloading injury and/or eccentric contraction injury of atrophied muscle) may have contributed to this disparity. Our evidence to date suggests that transgenic over-expression of IGF-1 in skeletal muscle, which can induce junctional alterations in some systems and is proposed as a potential countermeasure for unloading-induced muscle atrophy, does not exacerbate junctional alterations in this model system. Finally, our preliminary data indicating alteration of intracellular calcium and of calcium-dependent processes within motoneuron terminals suggest the possibility of increasing calcium-binding protein expression as a potential countermeasure for the observed alterations of neuromuscular junctions with hindlimb unloading. Our comprehensive approach using electrophysiologic and ultrastructural techniques is being extended to determine the junctional effects and tolerability of transgenic overexpression of a calcium-binding protein, parvalbumin, and to determine whether parvalbumin overexpression can ameliorate motoneuron dysfunction and/or muscle atrophy in mouse models of muscle atrophy and of neuromuscular diseases. Data obtained from this study will be useful in defining the anatomic and physiologic consequences to motoneurons of manipulations which induce muscle atrophy, and will aid in designing further experiments to determine the mechanisms influencing motor unit dysfunction occurring during space travel. Information from this study will be of value to the design and refinement of countermeasures aimed at ameliorating the deleterious effects of microgravity on human motor performance. The results of this work may also provide new insights into important clinical problems such as mechanisms influencing motoneuron dysfunction in devastating degenerative illnesses such as amyotrophic lateral sclerosis, muscle and motor nerve injury encountered in critical care settings, and the design of therapies to retard or prevent muscle atrophy produced by disuse or spinal cord injury.
Functional and structural alterations at the NMJ with hindlimb unloading In several experimental series, a 3-week period of hindlimb unloading (HU) by tail suspension was well tolerated and produced robust effects on muscle atrophy, with atrophy in the soleus (SOL) muscle consistently exceeding atrophy of more mixed muscles such as the plantaris and gastrocnemius. Small differences attributed to strain or age of mice were noted across experimental groups. Effects of HU remained statistically significant if corrected for body weight changes, except for changes in the plantaris. These effects are similar to changes reported previously (Criswell et al., 1998). Longer term suspensions (up to 6 weeks) have not resulted in appreciable increases in the degree of muscle atrophy (data not shown). In muscles acutely dissected from 2-3 month ICR mice and studied with intracellular recording techniques in normal calcium saline, spontaneous transmitter release, as estimated by miniature end-plate potential (MEPP) frequency, did not differ significantly in soleus (SOL) muscles of control and 3-week HU mice, despite presence of severe atrophy in this muscle. In striking contrast to the findings from the SOL, significant increases in MEPP frequency were consistently recorded from the plantaris (PLT) following a 3-week period of HU. These changes were noted in multiple strains (FVB, ICR, BALB/c) and ages of mice (ranging from 2 to 10 months of age; data not shown). Values for other parameters (recorded ex vivo while maintaining the muscle at its minimal in situ length; see below), such as muscle fiber resting membrane potential and MEPP amplitude, did not differ consistently among groups, except for a slight decrease in resting membrane potential in soleus muscle fibers, perhaps resulting from effects of atrophy. These data suggest an effect of HU on motoneuron terminals, a presynaptic locus of action, and are consistent with the hypothesis that inactivity-induced alterations (perhaps retrograde signals from inactive muscle) result in modification of neuromuscular junction (NMJ) function. Further studies using longer-term (~6 week) suspensions indicate that with continued unloading, MEPP frequency changes may return to near control levels in the PLT muscle, while actually increasing slightly (~20%; p < 0.01) in the SOL. These data highlight the importance of defining the time course of the observed changes, a key part of the work proposed. To correlate the observed changes in transmitter release with ultrastructural changes at NMJs, we transcardially perfused ICR mice (control vs. HU for 3 weeks) following excision of muscles for ex vivo physiologic studies. A striking change in the ratio of postsynaptic to presynaptic membrane surface of randomly selected terminal boutons, evident on nearly every pair of micrographs examined, was present in the SOL muscle. As motoneuron terminal bouton size did not appear to change in this study, and the number of junctional folds appeared to be clearly reduced, this change was interpreted as a postsynaptic alteration resulting from unloading. This reduction in the post:pre surface ratio was approximately 35%, comparable to the degree of whole muscle atrophy in the SOL when adjusted for body weight changes with HU. We did not observe significant changes in the PLT with respect to surface ratios, or in either muscle with respect to Schwann cell envelopment of the terminal (a sensitive indicator of denervation in our previous studies [Siklos et al., 1996] as well as in the literature), synaptic vesicle density, density of vesicles around active zones (not shown), or mitochondria in the nerve terminal. Very few NMJs with junctional folds denuded of overlying terminals or enveloped by Schwann cells, and none with prominent presynaptic alterations, were noted in these assays, in contrast to the findings reported in space-flown animals (e.g., Riley et al., 1990a, Babakova et al., 1992; D'Amelio & Daunton, 1992). As we do not believe that this discrepancy can be explained solely by differences in sampling technique, we hypothesize that differences among the models (e.g., the effects of reloading injury in atrophied muscle) may contribute to the higher incidence of abnormal NMJs observed in space-flown animals. Tests of this hypothesis form the basis for future proposed work. To better assess the overall function of the NMJ in muscles of mice subjected to HU, we developed and validated the technique of stimulated single-fiber electro- myography (S-SFEMG) for use in the mouse gastrocnemius (Gooch & Mosier, 2001). S-SFEMG jitter, measured as the mean consecutive difference (MCD) in latency between successive single muscle fiber action potentials evoked by nerve stimulation, correlates well with the safety factor for neuromuscular transmission (e.g., Lin & Cheng, 1998). Following 3 weeks of HU in anesthetized ICR mice, we observed, at low rates of stimulation (2/sec), a striking increase in mean jitter values (by ~200%) in single fibers of unloaded gastrocnemius muscle, suggesting insecure neuromuscular transmission. Although actual blocking (failure) of transmission was noted in some fibers in this study, we would caution against directly extrapolating these results to unanesthetized human subjects. However, these results raise the possibility that, especially under predisposing conditions (e.g., drug effects, thermal injury, etc.), failure of neuromuscular transmission could become clinically significant in atrophied muscle. It is also worth noting that increases in jitter have been reported in cast-immobilized soleus muscles in human subjects (Grana et al., 1996), highlighting the potential importance of the present findings. As previously noted, what is not known is the time course of unloading-induced alterations in NMJs, which could vary among muscles due to differences in a number of factors, including fiber type distribution, mechanical stretch, and patterns of activation in the unloaded state. Furthermore, a precise correspondence between functional and ultrastructural changes with unloading remains to be established (Reviewers please note: an apparent mismatch between functional changes and structural alterations at NMJs has been reported by others [see Colman et al., 1997; Prakash et al., 1999], which may reflect the order of causation, or differences between underlying mechanisms). The time course measurements and correlative studies proposed are designed to quantitatively address these questions, as well as better defining mechanisms underlying the striking changes in NMJ function associated with muscle unloading. Muscle-motoneuron interactions at the neuromuscular junction We examined the effects of muscle stretch on spontaneous transmitter release in hindlimb muscles from unloaded mice, both to rule out a potential confounding variable for the MEPP frequency studies above, and to investigate the possibility of a potential mechanical or contact-mediated muscle-motoneuron interaction which could alter NMJ physiology with unloading. In general, the mammalian skeletal NMJ is insensitive to muscle stretch. Our findings in SOL and PLT, based on ex vivo stretch from the minimal muscle length measured in situ (comparable to the ex vivo relaxed length) to the maximal length measured in situ, are consistent with these reports, with no significant effects of muscle stretch on MEPP frequency observed in either muscle (data not shown). However, in the EDL muscle, which by our measurements is always held in a slight amount of tension (minimal in situ length exceeding fully relaxed length by ~5%), we noted a decrease of MEPP frequency with unloading, and a further reduction of MEPP frequency with muscle stretch within the physiologic range, with no apparent effect on resting membrane potential or MEPP amplitude among any of the tested groups. These data suggest that muscle stretch effects, at least acutely, do not account for the HU-associated changes in spontaneous release in the posterior compartment muscles (SOL and PLT) tested above. However, the data raise the possibility that a small degree of stretch sensitivity of release may become apparent in a mixed muscle of the anterior compartment (EDL), and studies of NMJ function with HU in this muscle must take into account the potential effects of mechanical influences. Whether these effects of muscle stretch are associated with changes in evoked release, or other alterations in the NMJ, is not known; nor are the NMJ effects of chronic, passive muscle stretch in vivo understood at the present time. [Reviewers please note: the small effects of muscle stretch on spontaneous release in these experiments should not be confused with the dramatic effects of muscle stretch on evoked release at amphibian NMJs, where enhancement of release is of sufficient magnitude to act as a peripheral amplifier of the stretch reflex (e.g., Chen & Grinnell, 1995). We believe that our data confirm previous observations of the general insensitivity of mammalian NMJs to stretch effects, but are of value in that they suggest caution in the interpretation of small differences in observed NMJ function where muscle length in the assay system is not controlled]. Changes in intracellular Ca2+ with hindlimb unloading In our ultrastructural studies of NMJs to date, we have not observed changes in the number of active zones, vesicle density around active zones, or evidence of sprouting to account for the observed increases in MEPP frequency in the PLT muscle with HU. An attractive candidate mechanism for increased spontaneous release in this model system is an increase in intracellular calcium concentration at the motoneuron terminal. A large body of published reports implicate intraterminal calcium as a key regulating signal for neurite extension, growth cone guidance and interaction with target muscle fibers, effects of muscle-derived trophic factors, and sprouting (for recent discussions see Boulanger & Poo, 1999; Graf et al., 1999; Lautermilch & Spitzer, 2000; Santafe et al., 2000). As an initial test of the hypothesis that increased intraterminal calcium contributes to the NMJ changes observed with HU, we employed electron microscopy to assay intracellular calcium using an oxalate- pyriantimonate precipitation technique (e.g., Siklos et al., 1996, 1998). Preliminary data from this approach (generated from 4 of an anticipated 10 pairs of mice) suggest that HU of 3 weeks' duration may be associated with a 44% increase in cytosolic Ca in motoneuron terminals of the PLT muscle, while producing little or no changes in terminals of the SOL muscle (-8%). These differences, while not statistically significant in this interim analysis of < 1/2 of the planned experiments, appear to parallel the changes in MEPP frequency observed with HU in the PLT and SOL muscles. Further work is needed to confirm these interim findings, and to better define the magnitude of the observed effects, and is presently in progress. These data, taken together with the extensive evidence implicating intracellular calcium as a key signal for neuromuscular junction formation and modification, suggest that intracellular calcium may be elevated in motoneuron terminals of muscles subjected to hindlimb unloading. Elevations in intracellular calcium, while not observed in studies to date to reach the levels believed to be associated with mitochondrial toxicity in disorders such as amyotrophic lateral sclerosis (Siklos et al., 1996), may be sufficient in NMJs of hindlimb-unloaded animals to act as physiological signals for junctional remodelling, and to induce significant alterations in presynaptic terminal function. The time course of these changes with unloading or reloading is presently unknown. It is also not known whether interventions known to stabilize neuronal Ca2+ levels in vivo, such as increased expression of the Ca2+-binding protein parvalbumin, can attenuate the NMJ alterations observed with muscle unloading. Calcium-binding protein overexpression, Ca2+ homeostasis, and neuroprotection in motoneurons To test the hypothesis that overexpression of Ca2+-binding proteins can modulate neuronal calcium perturbations and influence calcium-dependent processes in mice, we generated transgenic mice expressing rat parvalbumin (PV) under control of the rat calmodulin II (CaMII) promoter (Beers DR, et al., in preparation). Briefly, four founder mice were identified by Southern blots; of these, two (lines 12 and 14) were bred to homozygosity. RT-PCR detected high levels of rat PV transcripts in spinal cord, skeletal muscle, and liver; in situ hybridization with an antisense probe confirmed PV mRNA expression in lumbar, cervical, and hypoglossal motoneurons of PV transgenic lines, but not in control mice. PV mRNA expression was not observed in glial cells adjacent to motoneurons. Western blots confirmed PV protein overexpression in spinal cords of PV transgenic mice, to a level of ~5x controls. Quantitative determination of PV protein expression in skeletal muscle of PV transgenic mice is currently in progress. Immunohistochemistry confirmed significant PV protein expression in spinal cord and hypoglossal motoneurons of both transgenic lines, but little or no expression was detected in motoneurons of control mice. PV overexpression did not appear to alter the expression of another calcium-binding protein, calbindin-D28K, in motoneurons. Both PV-overexpressing lines are similar in weight and gross morphology to the B6/SJL background strain. In a small number of animals, we have observed no obvious changes in S-SFEMG jitter with parvalbumin overexpression (mean SD: 12 4 ms for PV line 14; 10 3 ms for wild type controls, p = 0.17), suggesting that the safety factor for overall neuromuscular transmission is not greatly altered with parvalbumin overexpression. We have not observed increases in susceptibility to hypoventilation with usual doses of anesthesia in the parvalbumin transgenic mice, again suggesting that no gross dysfunction of motor units occurs with parvalbumin overexpression. A trend toward enhanced Schwann cell envelopment of boutons was noted in PV transgenic mice with respect to controls (borderline significance, p = 0.05), while clear increases in the postsynaptic: presynaptic surface ratio were evident in PV transgenic mice (p < 0.001), suggesting the possibility that PV overexpression may influence NMJ remodelling. Denuded postsynaptic folds or altered vesicle density in terminals was not observed to an appreciable extent in control or PV transgenic mice. Although not quantified at present, the parvalbumin transgenic lines appear to exhibit similar levels of motor activity to controls, and do not exhibit spontaneous seizure activity on EEG screening (Noebels JL, pers. comm.). Thus, there is no evidence at present that gross locomotor activity or neuromuscular function are adversely affected by long-term transgenic overexpression of parvalbumin. To determine whether PV overexpression was associated with enhanced calcium homeostasis in motoneurons, we measured intracellular calcium (by oxalate- pyriantimonate precipitation) and spontaneous transmitter release (by intracellular recording) following in vivo passive transfer of sera from patients with ALS. This intervention, which causes elevations in intraterminal calcium and MEPP frequency at NMJs within 24 hours in vivo (Engelhardt et al., 1995; Mosier et al., 2000), produced robust effects in wild-type control mice, but failed to alter intracellular calcium or transmitter release in either line of PV transgenic mice (not shown). In vitro alteration of bath calcium over a log-unit range increased Ca2+-dependent spontaneous release to a much lesser extent in nerve-muscle preparations from PV transgenic mice than from controls. These data, taken together, suggest considerable attenuation of intracellular calcium increases and of changes in spontaneous release, a calcium-dependent process, in motoneuron terminals of parvalbumin-overexpressing mice. To test the hypothesis that parvalbumin overexpression in motoneurons confers protection against motoneuron injury, parvalbumin transgenic mice were crossbred with transgenic mice overexpressing the G93A mutation of human superoxide dismutase (SOD1), a mouse model of familial amyotrophic lateral sclerosis (ALS). In doubly transgenic mice overexpressing mutant SOD1 (G93A) and parvalbumin, highly significant increases in overall survival were observed, principally accounted for by a delay in the age of onset of clinical evidence of motoneuron disease. Preliminary data (D.R. Beers and S.H. Appel) suggest a trend toward improved motoneuron cell body survival in these doubly transgenic mice, compared to mutant SOD1 transgenic controls. These effects have persisted over ~8 generations, and are present in both transgenic lines, suggesting that differences in genetic background or site of transgene insertion are unlikely to be confounding factors. These encouraging results suggest that transgenic overexpression of parvalbumin is associated with clinical protection in a model of motoneuron injury, and may also be neuroprotective in these mutant SOD1 mice. Whether overexpression of parvalbumin, either in motoneurons or in skeletal muscle, can protect against muscle atrophy or NMJ alterations associated with unloading or reloading, is not known, and forms the basis for future proposed work. Summary. These results, taken together, suggest that structural and functional changes in the innervating motoneuron may accompany the process of muscle atrophy induced in a mouse model of hindlimb unloading (HU). It is apparent from the single-fiber EMG studies that such changes are associated with insecure neuromuscular transmission (increased "jitter" suggesting a reduced safety factor). These changes appear to be associated with evidence of altered intracellular calcium in motoneuron terminals. The nature and extent of these changes remains to be better defined by ongoing and planned electrophysiologic and ultrastructural studies, with time course series and interactions with reloading or eccentric contraction-induced injury being high priorities. Preliminary data indicate that IGF-1 overexpression in muscle of transgenic mice is unlikely to worsen the effect of H.U. on at least one aspect of NMJ function. Further studies will be necessary to conclusively rule out a deleterious effect of IGF-1, or to determine whether it has a beneficial effect in this model system. The characterization of mice overexpressing a calcium-binding protein, parvalbumin, in motoneurons and skeletal muscle, has made it possible to begin to pursue the goal of modifying calcium-binding protein expression as a potential countermeasure for motoneuron injury in motoneuron diseases such as ALS, and for muscle and/or neuromuscular junction alterations occurring with unloading-induced atrophy. Implications for Further Research. Whether the neuromuscular junction alterations described in this model have adaptive or detrimental significance (or possibly both) is not yet fully clear. Increases in transmitter release could help to compensate for deficits in motor unit function (as suggested for myasthenia or myasthenia-like syndromes), or to provide trophic influences on muscle function. However, if up-regulation of spontaneous release predisposes to synaptic depression at higher frequencies of stimulation, these same modifications could be detrimental to motor unit performance. Furthermore, if up-regulation of release from motoneurons innervating atrophied muscle is compensatory, it is possible that the mechanisms subserving this change could be highly vulnerable to inadvertent blockade (and consequent decrement in motor unit function) by certain medications. A precedent for this possibility has been hypothesized to account for the unusual sensitivity of patients with myasthenia gravis and other neuromuscular junction disorders to frequently employed medications such as ?-blockers, calcium channel antagonists, glucocorticoids (at either pharmacologic doses, or even at physiologic concentrations encountered under stress situations), and aminoglycoside antibiotics. Future studies will need to address these possibilities. Multiple mechanisms may mediate the unloading-induced NMJ alterations observed in this study. The presynaptic changes are hypothesized to result from a muscle activity-dependent, retrogradely acting factor such as IGF-1 or related signaling molecules (e.g., GDNF). Continuing experiments will begin to test these possibilities. The increases in spontaneous release observed in the plantaris could result from increased intracellular calcium, even in the absence of a clear morphologic alteration in vesicles or evidence of synaptic remodeling. These mechanisms are potential targets for therapeutic intervention. Altered neuromuscular junction structure and function could result in changes in neurally modulated aspects of muscle physiology, such as acetylcholine receptor (AChR) expression. Limb immobilization in rats has been shown to up-regulate AChR expression in skeletal muscle (Yanez et al., 1996), which could predispose to life-threatening hyperkalemia or to reduced responsiveness to neuromuscular blockers in the setting of a medical emergency requiring intubation. To date, no published reviews of the challenges of inflight surgical interventions have addressed this possibility. Muscle specimens have been collected from ongoing experiments with unloaded mice to begin to address this question, using 125I-a- bungarotoxin binding of AChRs. Impact on Critical Path Risks. The work performed has contributed toward understanding neuromuscular junction changes associated with spaceflight. Based on the results of this work, it is clear that neuromuscular transmission, while unlikely to fail in healthy, unloaded muscle, is insecure, and may prove susceptible to failure in the presence of other stresses likely to be encountered in spaceflight (e.g., hypothermia, reloading injury, and commonly administered medications). This would affect muscle performance in the areas of fatigue and loss of muscle power, which would lead to inability to perform specific tasks, and contribute to risk of injury. More research is needed to clarify these risks, and to determine which risks can be avoided by specific interventions such as exercise or pharmacologic interventions, and which risks can be avoided by minimizing exposure to stresses that could impair junctional transmission. Extensions of the Proposed Work. It is unclear whether the changes observed at the neuromuscular junction in this study also occur at other synapses (e.g., the Ia-to-alpha-motoneuron synapse subserving the monosynaptic stretch reflex). The potential involvement of altered intracellular calcium in this model suggests the possibility of testing countermeasures which could alter calcium handling in motoneurons, such as the vitamin D analogs, which are being tested in the laboratory of Dr, Stanley Appel (Alexianu et al., 1998). A transgenic mouse line overexpressing parvalbumin in neurons, developed in the laboratory of Dr. Appel, is presently being characterized electrophysiologically in my laboratory. It is of great interest that the vitamin D analog EB1089, which has been proposed to retard bone loss induced by microgravity, could also have effects on motoneuron or muscle calcium handling. It also remains to be tested whether the processes defined in the mouse HU model can be extended to human patients. In this regard, the stimulated single-fiber electromyography (SFEMG) studies which are currently being tested in this model have an extensive record of usefulness in the electrodiagnosis of human neuromuscular diseases, and could be immediately applied to testing of human subjects after prolonged bed rest or spaceflights of varying duration. The data obtained from this work are thus likely to be of immediate value in the design of studies aimed at defining the contribution of motoneuronal influences on muscle to overall motor unit function in humans exposed to spaceflight. Data from this work has also advanced our understanding of the impact of peripheral (muscle and neuromuscular junction) alterations on overall motor dysfunction in devastating human motoneuron diseases such as amyotrophic lateral sclerosis (ALS). Preliminary data generated in part by this NSBRI-sponsored work suggest the feasibility of altering calcium-binding protein expression as a potential countermeasure in patients suffering from ALS, a major area of interest for our laboratories.
Disuse atrophy of skeletal muscle is a significant clinical problem in human patients after cast immobilization, prolonged inactivity in the intensive care unit setting, or in neuromuscular disorders producing clinical weakness. By developing a mouse model of muscle unloading during spaceflight, many of the conditions leading to disuse atrophy of muscle in commonly encountered clinical situations may be simulated. Development of an animal model of neuromuscular junction changes occurring during unloading-induced atrophy could provide significant insights into the mechanisms underlying neuromuscular disorders with a component of neuromuscular junction or distal motoneuron dysfunction, including critical care neuropathies and amyotrophic lateral sclerosis. Furthermore, the countermeasures developed and tested in this work may have clinical benefit in these devastating human disorders, and one of the proposed countermeasures (IGF-1) is being evaluated at this center in human patients suffering from amyotrophic lateral sclerosis.
FY00 Publications, Presentations, and Other Accomplishments:
Mosier, D.R., Pang, J., Siklos, L., Beers, D.R., Gooch, C.L., and Appel,S.H. ''Enhanced parvalbumin expression attenuates motoneuron responses to calcium elevation.'' (abstract) Neurology, 54 (Suppl. 3), A305 , (2000). Mosier, D.R., Sikls, L. and Appel, S.H. ''Resistance of extraocular motoneuron terminals to effects of amyotrophic lateral sclerosis sera.'' Neurology, 54, p. 252-255 , (2000). Gooch, C.L. and Mosier, D.R. ''Stimulated single-fiber electromyography in the mouse: techniques and normative data.'' Muscle & Nerve , (2000). Appel, S.H., Alexianu, M.E., Engelhardt, J.I., Sikl s, L., Smith, R.G., Mosier, D. & Habib, M. ''Involvement of immune factors in motoneuron cell injury in amyotrophic lateral sclerosis, '' in ''Amyotrophic Lateral Sclerosis, p. 309- 326.'' ed. Brown, R.H. Jr, Meininger, V. & Swash, M., Martin Dunitz, London , 2000. Appel, S.H., Smith, R.G., Lai, E.C., Mosier, D.R. & Haverkamp, L.J. ''Amyotrophic lateral sclerosis'' in ''Prognosis of Neurological Disorders, 2nd ed., p. 513-519.'' ed. Evans, R.W., Baskin, D.S. & Yatsu, F.M., Oxford University Press, Oxford, 2000. Mosier, D.R. ''Basic neurophysiology of the presynaptic terminal: modifiability in normal and disease states.'' Single Fiber EMG Special Interest Group, American Academy of Electrodiagnostic Medicine 47th annual meeting (Sept. 16, 2000).

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