Recovery of rat tibialis anterior motor unit properties following partial denervation GORDONJ. BELL,KATHYAYER, ~ S S GORDON, A SURESHDEVASHAYAM, A N D THOMASP. MARTIN) Division of Neuroscience, Deparlments of Physical Education and Sporb Studies, P~tarmucolog~~, and Physical Tkerapy, Us.lkvers.s&@ of Alberta, Edmonton, Aka., Calaada T66 2G4
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Received April 6, 1992 BELL.6. J., AYER,K., GORDON,T., DEVASHAYAM, S., and A ART IN, T. P. 1992. Recovery of rat tibialis anterior motor unit properties following partial denervation. Can. J . Physiol. Pharmacol. 78: 1324-1329. The recovery of selected mechanical, morpho8ogical, and metabolic properties of rat tibialis anterior fast motor units was determined following partial denervation (n = 7) or partial denervation and hemispinal cord transection (8s = 5) and compared with age-matched control units (n = 7). Following 1 - 12 months of recovery9 the mechanical properties of each unit were measured and the fibres depleted of glycogen by using standard ventral root filament stimulation techniques. Quantitative histohemical techniques were used to determine cross-sectional area and the activities of succinate dehydrogenase and a-glycerolphosphate dehydrogenase in individual unit fibres. Partial denervation increased the mean fibre area but decreased a-glycerolphosphate dehydrogenase activity. Succinate dehydrogenase was unchanged in the denervated groups. The variability in area and enzymatic activities among the unit fibres was urachanged. However, the interrelationship between the enzymes was altered by both denervation procedures. Succinate dehydrogenase activity was directly related to fatigue resistance and inversely related to tetanic tension across the units. These findings suggest that a motor unit reestablishes many of its properties despite marked changes to the composition of the unit brought about by partial denervation. In addition, a reduction in the neuromuscular activity of units during reorganization had a limited effect on recovery. Key words: motor unit, succinate dehydrogenase, a-glycerolphosphate dehydrogenase, spinal cord hemisection. BELL,6. J., AYEW,K., GORDON,T., DEVA~HAYA~I, S., et MARTIN,T. 1992. Recovery of rat tibialis anterior motor unit properties following partial denervation. Can. J . Physiol. Pharmacol. 78 : 1324- 1329. On a determint le rCtablissement de propriCtCs mCtaboliques, n~orphologiqueset mkcaniques sp5cifiques d'unitts motrices rapides du muscle jambier anttrieur de rat aprks une dknervation partielle (n = 7), ou aprks une dCnervation partielle et une hknaisection de la moelle (n = 5 ) , et 19avonscompare au rCbblissement d'unitks tCmoins de rats du mCme 2ge (n = 7). Aprks 1 - 12 mois de retablissement, on a mesurd Ies proprietts mecaniques de chaque unite et appauvri Bes fibres en glycogene, en utilisant des techniques standard de stimulation de filaments de la racine ventrale. On a ueilisC des mkthodes hjistochimiques quantitatives pour determiner la section transverse et Les activites de succinate-dCshydrogCnase et d'a-glyc6ralphosphate dCshydrogCnase dans des fibres individuelles. La dknervation partielle a augment6 la surface moyenne des fibres, mais a diminuC l'activitk d'a-glyc6rolphosphate deshydrogtnase. La succinate dCshydrogCnase des groupes dknervks n'a pas variC. La variabilite dans les activitks enzymatiques et superficielles interfibres est demeuree inchangee. Toutefois. la relation entre les enzymes a Ctt aleCree par les deux modes de dinervation. L9activitC de sucsinate dkshydrogtnase fibrillaire a Ctk relliCe directement a la rCsistance h la fatigue et inversement & la tension tetanique. Ces rksultats suggkrent qu'une unit6 motrice ricupkre plusieurs de ses proprittCs malgrt des variations rnarqukes de la composition de 1'unitC provoqukes par la dknervation partielle. Be plus, une rkduction de I'activitt neuromusculaire des unites durant Ia rtorganisation a eu urn effet limit6 sur le retablissement. Mobs c l b : unite motrice, succinate dCshydrogCnase, or-glycCrolphosphate dCshydrogCnase, hdmisection de la rnoele. [Traduit par la rkdaction]
Introduction Following the partial denervation of skeletal muscle, the remaining intact motor units have the ability to enlarge their peripheral fields and compensate for the lost motoneurons (Brown et a&.1981; Brown and Hronton 1987; Kugelberg et a&. 1970; Luff et al. 1988; Luff and Torkko 1990; Tissenbaum and Parry 1991). This compensatory mechanism may be limited in that there appears to be a maximal capacity for neuronal sprouting (Brown et a&. 1981 ; Brown and Ironton 1987; Luff et al 1988; Luff and Torkko 1990). It has been suggested that there is a relationship between sprouting capacity and the efficacy of the sprouts with developmental age (Jacob and Robbins 1990; Lswrie ek a%. 1998; Rosenheirner 1990). In addition, neural and muscular activity have been reported to have some (Connold and Vrbova 1990, 1991; Ribchester and Taxt 1984) or no (Gardiner and Faltus 1986) impact on sprouting capacity. Partial denervation provides a rigorous test of the ability of a motor unit to recover its properties since the complement of fibres comprising a unit is altered. Despite the considerable [Author for correspondence, at the Department of Physical Therapy. Prlntcd
In
Canada / lmpr~mCau Canada
amount of information available concerning a muscle's response to partial denervation, the ability of individual units to reestablish their mechanical, morphological, and metabolic properties remains undefined. In addition, the role of neurombascular activity in the recovery from partial denervation is unclear. Therefore, this study was undertaken to determine the ability of rat tibidis anterior units to recover their mechanical and metabolic properties following partial denervation alone or in conjunction with hemispinal cord transection induced paralysis. Our observations suggest that the recovery of rat tibialis anterior fast units following partial denenation is rather robust. Furthermore, the reduction in neuromuscular activity (i .e. , hemispinalization) had only a limited impact on recovery.
Materials and methods Punial denemtion and spinal herntsectkosa All animal experimentation was conducted according to Canadian and University of Alberta guidelines for animal research. Twelve female Sprague - Dawley rats (220 -260 g body weight) were anesthetized (i.p., sodium pentobarbitone, 35 mg/kg) and the right ventral roots L3, U,and L5 were exposed under sterile conditions. To achieve varying degrees of partial denervation (PD group) of the tibialis anterior
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BELL ET AE.
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(TA), either one or two of the roots were cut and a portion of the distal aspect of the root(s) was excised. A hemisection of the spinal cord at T12 also was performed in five rats (HPD group). Subsequently, the animals were allowed to recover for 1 - 12 months. Seven additional age-rnatched control rats (no partial denervation or hemisection) were maintained under similar conditions.
irurtninul experirnentation The water given to each rat was replaced with a 5 % glucose solution to augment the glycogen levels of the TA 48 h prior to terminal experimentation. The rats were prepared for the isolation of single ventral root filaments under anesthesia using standard techniques (Gillespie et al. 1987). Briefly, a laminectomy was performed to expose the spinal cord from T13 to L6. The sciatic nerve and TA from both sides were dissected and all other hindlimb and hip muscles denervated. An EMG electrode was sewn into the fascia of the TA and a pair of fine wires were embedded in the musculature immediately surrounding the sciatic nerve, allowing indirect stimulation of the TA. The rat was mounted in a rigid frame with the legs held stationary by clamps. The distal tendon was attached to a force transducer with the skin surrounding the leg and spinal cord used to create mineral oil pools. A heating pad and radiant heat were used to maintain core and muscle temperature at 35 -37°C throughout the experiment. Determinatiora qf degree qf parria/ denemation The twitch and tetanic tensions of the right and left TA were determined by stimulation of the respective sciatic nerves. Subsequently, the twitch and tetanic tensions recorded from the stimulation of ventral roots L3, L4,or L5 were determined. If any force was measured in the previously cut right-side root, this was taken as evidence for regeneration of that root and the data from that animal were not included in the present data. A comparison of the force contributions between the right and left sides of the respective roots (the sums of L3, L4, and L5 of the left versus right multiplied by 100) provided ran estimation s f the degree of partial denervation (%PD) of the right TA. Single motor unit isolation and g!ycogen depletion A single unit was isolated using standard ventral root filament splitting techniques (Gillespie et a / . 1987). This unit was analyzed for tetanic force (200-ms trains with a 18-ms interpulse interval), fatigue resistance (188 pulses/s in 58-ms trains repeated each second for 2 min, with the ratio of the tension at 2 min relative to the initial tension providing a fatigue index), and sag during an unfused tetanus (800-n~strains with an interpulse interval 1.25 times the twitch contraction time). These mechanical properties were used to classify the units according to the criteria of Burke eF al. (1973) as fast fatigable, fast fatigue intermediate, or fast fatigue resistant. Subsequently, the unit was stimulated using intermittent trains of 5 pulses with a 10-ms pulse interval to deplete the fibres of glycogen. Tension was recorded throughout the stimulation and, if the decline in tension reached a steady state, the train rate was increased until tension continued to decline. Stimulation was maintained until the tension failed to recover following a brief rest period or until the stimulation response was lost. Muscle preparation and quantitative histoehemistry Following the glycogen depletion procedures, the TA was removed, weighed, and cut into blocks, which were mounted on cork and frozen in isopentane cooled in liquid nitrogen. Tissue sections (20 prn thick) were cut transversely from each block and prepared for the determination of glycogen using a periodic acid - Schiff's reaction (PAS). The tissue area that yielded the region with the greatest density of unit fibres depleted of glycogen was further analyzed using quantitative histochemical techniques described in detail elsewhere (Martin et al. 19880, l988b). Briefly, the PAS section was digitized using a computer-assisted image analysis system (PSICOM 232, Perceptive Systems. Inc., League City, Tex.). The optical densities (ODs) of a random sample of glycogen-depleted fibres (mean n = 17, range I10 - 33) were compared with the densities of fibres that did not appear to be depleted of glycogen (mean n = 32, range 20-48). 1%the distribution of ODs was not overlapping, those fibres with low
TABLE1. Summary of partial denervation and recovery procedures Animal
Roots cut for partial denervation
Root location of depleted unit
PD recovery (days)
Control I 2 3 4 5
6 7 PD 1 2 3 4 5 6 7 HPD 1 2 3 4 5
OD were considered to be part of the isolated muscle unit, while the remaining fibres (high OD) were taken to be representative of nonunit fibres. The failure of this analysis to generate two distinct distributions of different OD resulted in the exclusion of the muscle from further analysis. Some units with mechanical properties consistent with the slow fatigue resistant type were identified in the TA. However, none were successfully glycogen depleted based on the OD distribution in the PAS-prepared tissue sections. Therefore, the present findings are restricted to the fast unit populations of the rat TA. As a result. the nonunit fibres analyzed were also restricted to those that demonstrated a dark staining intensity (presumably from fast units) in the tissue section prepared for the qualitative assessment of myofibrillar ATBase (preincubation pH 8.8). In the muscles that contained a successfully isolated unit, serial tissue sections were cut and prepared for the determination of succinate dehydrogenase activity (SDH) (Blanco eb al. 1988), a-glycerolphssphate dehydrogenase activity (aGPD)(Martin et a / . 1988a), and a qualitative index of mysfibrillar ATPase staining intensity under alkaline (pH 8.8) preincubation conditions (Nwoye et a / . 1982). The same fibres were readily identified in the serial sections using the template of unit and nonunit fibres defined in the PAS section.
Data analysis Standard descriptive statistical procedures and linear regression analyses using Bearson's correlation coefficient were conducted using the PC-based statistical software program STATGRAPHICS (STSC, Inc., Rockville, Md.). Differences in dependent variables between groups and muscle units were analyzed using a two-way analysis of variance (ANOVA) and significant main effects and (or) interactions were analyzed using a Newman-Keuls multiple comparison procedure. Results were considered statistically significant if p < 0.05.
Results All successfully glycogen depleted units were classified according to their mechanical properties as fast fatigable, fast fatigue resistant, or fast fatigue intermediate. The sectioning of roots L3, L4, or L5 singularly, or in combination, yielded 2 - 80% partial denervation (Tables 1 and 2). Similar procedures in the presence of spinal cord hemisection resulted in
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CAK. J . PHYSIOL. PMAWMACOL. VOL. 90, 1992
TABLE2. Properties of the tibialis anterior motor units
Condition
Tetanic Fatigue tension %PD index (mN)
SDH (OD x 103/min), mean SD
Fibre area (p2). mean f SD Unit (CV)
+
Nonunit (CV)
Unit (CV)
Nonunit (CV)
GPD (OD x IO"/min), mean SD
+
Unit (CV)
Nonunit (CV)
Control
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1
2 3 4 5 6 7 PD 1 2 3 4 5 6 7
HPD 1 2 3 4 5
8-98% partial denervation. It should be noted that partial denervation above approximately 75 % resulted in the incomplete recovery of whole muscle force, suggesting a limit of sprouting potential in the rat TA. Table 2 provides the tetanic-tension and fatigue-resistance properties of the units; fibre area; and SBH (see d s o Fig. I) and GPD activity for the unit and nonunit fibres. There was a significant main effect for fibre area that resulted in a significant difference in the coefficient of variation (CV) between the motor unit and non-motor-unit fibers. A significant interaction effect revealed that the CV for fibre area was significantly different between motor unit and non-motor-unit fibres for both the control and partial denervated (PD) group but not the hemisected (HPD) group. Also, the CV for fibre area in the control nsnmotor unit was significantly different from the HPD nonmstor unit. There was a significant main effect for the CV of SBH and GPD activity. Thus, the CV of SDH and GPD activity was lower in the motor unit compared with the nonmotor units ( p < 0.05). No sther statistical differences were observed. The activities of SDH and GPD were found to be inversely related within the fibres of the control motor units (r = 0.$6, p < 0.05; Fig. 2). This relationship was absent in both the PD and HPD groups. Because SDH activity was unchanged Crelative to the control) in these latter groups, the decrease in GPD in the PD group and the elevated GPD in the HPD group may account for the differences. Mean SDH activity was positively related to fatigue resistance (Fig. 3). However, the relationship between fatigue resistance and SDH activity was only significant in the HPD units ( r = 0.98, p < 0.05). Finally, SDH activity was inversely related to tetanic tension but only achieved statistical significance in the control motor units (r = 0.78, p < 0.05; Fig. 4). One motor unit in the PD group displayed an inordinately high tetanic tension and SDH activity that could not be explained and was excluded from the analy-
sis. In general, the oppsiste relationship existed between the mechanical properties and GPD activity by virtue of the inverse relationship between GPD and SHD activities within the motor units.
It has been estabIished that in response to partial denervation the intact motoneurons enlarge their peripheral fields through collateral sprouting and reinnervate the denervated fibres (Brown et al. 1981; Brown and Ironton 1987; Kugelberg et a!. 1970; Luff et &a[. 1988; Luff and Torkko 1990; Tissenbaum and Parry 1991). Consequently, following sprouting, motor units are composed of fibres that previously belonged to disparate units with potentially very different properties. As a result, partial denervation provides an opportunity to investigate the strength sf the interrelationships among the properties of a unit. Previously, we reported that a considerable degree of variability in fibre size and metabolic enzyme activities existed within units of the cat soleus (Martin et al. 1988w) and tibilais anterior (Martin et a!. 19886). Although these findings are corroborated by the present data, as well as by other studies (Edstrom and KugeBberg 1968; Enad et a / . 1989), they are contrary to data that suggest that a unit may be composed of fibres with essentially identical properties (Nerneth et wl. 1981, 1986). Despite differences in the literature as te, the degree of the variability among the fibres of a unit, it is clear that the variance within a unit is significantly less than that found across a sample of different units in the same muscle. Therefore, we affirm our interpretation of these data to suggest that the properties s f a motor unit are regulated at the level of the "unit type" rather than at the level defined by a unit with fibres having identical properties (for further discussion see Martin et al. 1988a, 1988h). Some of the PI3 motonebarons enlarged their peripheral
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BELL ET AL.
Control Unit
PD Unit
HPD Unit
Control nonunit
P D nonunit
HPD wonunit
SDH
(OD/min, ~ 1 0 0 0 )
SDH. (8D/min, XI 000)
SBH
(BD/min, xdOOQ)
FIG.1. Frequency distributions of SDH activity for motor unit (open bars) and non-motor unit (solid bars) fibres from control (No. l), PD (No. $1, and HPD (No. 3).
fields by as much as 4-fold despite no change in the variability of fibre size or SDH and GPD activity within a unit. %tis likely that at least some of the fibres analyzed in these units originated from different units prior to the partial denervation. This
suggests that the factor(§) responsible for the regulation of unit properties is particularly puissant. It has been reported that motor unit fibres, after only 7 days of partial denervation, appeared to be more variable in size and SDH histochemical
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CAN. J . PHYSIOL. PHARMACQBL. VQL. 70, 1992
6 0
326 484 SDH Activity (OD x 10 )
1 60
640
800
FEG.2. The relationship between the mean activities of SDH and GPD from control (9, continuous rule), PD (u,dotted rule), and HPB (n, broken line) units. The enzymatic activities were inversely related in the control ( r = -0.05, ns) and HPB ( r = -0.39, ns) units.
SDH Activity (08 x 8 $1
FIG. 3. The relationship between mean SDH activity and fatigue index of the units. These properties were linearly related in the control (B. = 0.56, ns), PD (B. = 8.62, ns), and HPD(r = 0.98, p < 0.05) units. Symbols are as defined in Fig. 2.
staining intensity relative to controls (Gardiner et al. 1987). Therefore, fibres in partially denervated units may proceed through a phase of reorganization that alters the properties of the new fibres to those defined by the confines of the original composition of the unit. It is tempting to conclude that the innervating rnotoneuron may be responsible for the regulation of the fibre properties within the unit. However, motoneuronal control may not act independently since only SDH activity in the HPD units appeared to be regulated comparably with the control and PD units. The variability in fibre size and GPD activity across nonunit fibres in the HPD muscles was essentially equal to that
FIG. 4. The relationship between mean SDH activity and tetanic force of the units. These properties were inversely related in the control (r = -0.78, p < 0.05) and HPD (r = -0.73, ns) units. With the exception s f one unit in the PD group, a similar relationship existed (P = -0.47, ns). The regression line for the P B group is drawn with this unit excluded (in parentheses). Symbols are as defined in Fig. 2.
within a single unit, suggesting that muscular activity and load may be important regulatory factors. A similar conelusion has been drawn from the study of partial denervation in the presence of bungarotsxin-induced paralysis (Csnnold and Vrbova 1990, 1991). Furthermore, the reduced variability in fibre area and CPD activity across nonunit fibres in the HPD group (but not within a unit) could also be related to a shift in the entire motor unit population toward a more homogeneous population. This has been suggested to occur in the cat soleus following spinal cord transection (Cope et ak. 1986; Martin et al. 1 9 8 8 ~ ) . The lack sf an inverse relationship between SDM and GPD activities in the PD and HPD units appeared to be primarily a result of changes in GPD activity rather than SDH. Whether the change in CPD activity was secondary to the respective perturbations or reflects a lack of complete recovery by the motor units was unclear. The former possibility would be consistent with reports of an overall increase in GPD activity following spinal cord transection in cat soleus units (Martin et al. 1 9 8 8 ~ and ) medial gastrocnemius fibres (Jiang et a!. 1990). The general maintenance of the interrelationships among SDH activity, tetanic tension, and fatigue resistance in the PD and HPD units illustrates another level of control over the properties of a unit. Tetanic tensions increased in both groups of partially denervated motor units, while SDM activity remained similar to control levels, suggesting that the largest units prior to sprouting (with lower SDM activities) remained the largest after sprouting. The range in fatigue resistance across rat plantaris muscle units has been reported to decrease following partial denervation (Gardiner and Faltus 1986). These authors suggested that this change was due to the loss of units characterized by the two extremes of fatigability. The present study could not address this point owing to the lack of data from units classifiable as slow fatigue resistance. However, it is clear that partial denervation alone or in the presence
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BELL ET AL.
of hemispinal transection did not alter the fatigue resistance (or SDH activity) of the units. In summary, our data suggest that mstor units are capable of rather precise recovery s f their properties in the face of partial denervation (up to approximately 75 %). Consequently, a dynamic range of whole muscle function should remain following the recovery period. The recovery of some properties is compromised when muscular activity and load are reduced during the restoration period. However. the impact of this added perturbation was of limited consequence to the functional indices of recovery.
This work was funded in part by the Rick Hansen Man in Motion Legacy Fund and the Alberta Heritage Foundation for Medical Research. The technical assistance of Neil Tyreman was greatly appreciated. BIanco, C. E., Sieck, G . C.. and Edgerton, V. R. 1988. Quantitative histochemical determination of succinic dehydrogenase activity in skeletal muscle fibres. Histochem. J. 28: 230 -243. Brown, M. C., and Ironton, R. 1987. Sprouting and regression of neuromuscular synapses in partially denervated mammalian muscles. J . Physiol . (London), 278: 325 - 348. Brown, M. C., Holland, R. L., and Hopkins, W. G. 1981. Motor nerve sprouting. Annu. Rev. Neurosci. 4: 17 -42. Burke, R. E., Levine, D. N., Rsairis, P., and Zajac, F. E. 1973. Physiological types and histochemical profiles in motor units of cat gastrocnemius. 9. Physiol. (London), 234: 732 - 748. Connold, A. L., and Vrbova, G. 1990. The effect of muscle activity on motor unit size in partially denervated rat soleus muscles. Neuroscience, 34: 525 -532. Connold, A. L., and Vrbova, G. 1991. Temporary loss of activity prevents the increase of motor unit size in partially denervated rat soleus muscles. J. Physiol. (London), 434: 107- 119. Cope, T. C., Bodine, S. C . , Fournier, M.. and Edgerton, V. R. 1986. Soleus motor units in chronic spinal transected cats: physiological and morphological alterations. J. Neurophysiol. 55: 1202 1220. Edstrom, L., and Kugelberg, E. 1968. Histochemical composition, distribution sf fibers, and fatigability of single motor units. J. Neurol. Neurosurg. Psychiatry, 31: 424 -433. Enad. J. G., Fournier, M., and Sieck, G. C. 1989. Oxidative capacity and capillary density of diaphragm motor units. J. Appl. Physiol. 67: 620 - 627. Gardiner. P. F., and Faltus, R. E. 1986. Contractile responses of rat plantaris muscles following partial denervation, and the influence of daily exercise. Pfluegers Arch. 406: 51 -56. Gardiner, P., Michel, R., Olha, A., and Pettigrew, F. 1987. Force
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and fatiguability of sprouting motor units in partially denervated rat plantaris. Exp. Brain Res. 66: 597-606. Gillespie, J. M., Gordon, T., and Murphy, P. R. 1987. Motor units and histochemistry in rat lateral gastrocnemius and ssleus muscles: evidence for dissociation of physiological and histochemical properties after reinnervation. J . Neurophysiol. 57: 92 1-937. Jacob, J. M., and Robbins, N. 1990. Differential effects of age on transmission in partially denervated mouse muscle. J . Neurosci . 10: 1522- 1529. Jiang. B., Roy, R. R., and Edgerton, V. R. 1990. Enzymatic plasticity of medial gastrocnemius fibers in the adult chronic spinal cat. Am. J. Physiol. 259: 507 - 5 14. Kugelberg, E., Edstrom, L., and Abbruzzese, M. 1970. Mapping of motor units in experimentally reinnervated rat muscle. J. Neurol. Neurosurg. Psychiatry, 33: 3 19 -329. Lowrie, M. B., Shahani, U . , and Vrbova, G. 19%. Impairment of developing fast muscles after nerve injury in the rat depends upon the period of denervation. J. Neurol. Sci. 99: 249-258. Luff, A. R.. and Torkko, K. 1990. Long-term persistence of enlarged motor units in partially denervated hindlirnb muscles of cats. J . Neurophysiol. 64: 1261- 1269. Luff, A. R., Hatcher, D. D., and Toskko, K. 1988. Enlarged motor units resulting from partial denervation of cat hindlimb muscles. J. Neurophysiol. 59: 1377 - 1399. Martin, T. P., Bodine-Fowler, S. C., and Edgerton, V. R. 1 9 8 8 ~ . Coordination of electromechanical and metabolic properties of cat soleus mstor units. Am. J. Physiol. 255: 684 -693. Martin. T. P., Bodine-Fowler, S. C., Roy, R. R., Eldred. E. E., and Edgerton, V. R. 1988b. Metabolic and fiber size properties of cat tibialis anterior motor units. Am. J. Physiol. 255: 43 -50. Nemeth, P. M., Pette, D., and Vrbova, G. 1981. Comparison of enzyme activities among single muscle fibres within defined motor units. J. Physiol. (London), 311: 489 -495. Nemeth, P. M., Solanki, L., Gordon, D. A., Hamm, T. M., Reinking, R. M., and Stuart, D. G. 1986. Uniformity of metabolic enzymes within individual motor units. J. Neurosci. 6: $92 - 898. Nwoye. L., Mommaerts, W. F. H. M., Simpson, D. R., Serayderian, K., and Marusich, M. 1982. Evidence for a direct action of thyroid hormone in specifying muscle properties. Am. J. Physiol. 242: 401 -408. Ribchester, R. R., and Taxt, T. 1984. Repression of inactive motor nerve terminals in a partially denervated rat muscle after regeneration of active motor axons. J. Physiol. (London), 347: 497 - 5 11. Rosenheirner, J. L. 1990. Ultraterminal sprouting in innervated and partially denervated adult and aged rat muscle. Neuroscience. 38: 763 - 770. Tissenbaum, H. A., and Parry, D. J. 1991. The effect of partial denervation of tibialis anterior (TA) muscle on the number and size of motoneurons in TA motornaacleus of normal and dystrophic (C57BL dy2-'ldy2J) mice. Can. J. Physiol. Pharmacol. 69: 17691773.
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Received April 6, 1992 BELL.6. J., AYER,K., GORDON,T., DEVASHAYAM, S., and A ART IN, T. P. 1992. Recovery of rat tibialis anterior motor unit properties following partial denervation. Can. J . Physiol. Pharmacol. 78: 1324-1329. The recovery of selected mechanical, morpho8ogical, and metabolic properties of rat tibialis anterior fast motor units was determined following partial denervation (n = 7) or partial denervation and hemispinal cord transection (8s = 5) and compared with age-matched control units (n = 7). Following 1 - 12 months of recovery9 the mechanical properties of each unit were measured and the fibres depleted of glycogen by using standard ventral root filament stimulation techniques. Quantitative histohemical techniques were used to determine cross-sectional area and the activities of succinate dehydrogenase and a-glycerolphosphate dehydrogenase in individual unit fibres. Partial denervation increased the mean fibre area but decreased a-glycerolphosphate dehydrogenase activity. Succinate dehydrogenase was unchanged in the denervated groups. The variability in area and enzymatic activities among the unit fibres was urachanged. However, the interrelationship between the enzymes was altered by both denervation procedures. Succinate dehydrogenase activity was directly related to fatigue resistance and inversely related to tetanic tension across the units. These findings suggest that a motor unit reestablishes many of its properties despite marked changes to the composition of the unit brought about by partial denervation. In addition, a reduction in the neuromuscular activity of units during reorganization had a limited effect on recovery. Key words: motor unit, succinate dehydrogenase, a-glycerolphosphate dehydrogenase, spinal cord hemisection. BELL,6. J., AYEW,K., GORDON,T., DEVA~HAYA~I, S., et MARTIN,T. 1992. Recovery of rat tibialis anterior motor unit properties following partial denervation. Can. J . Physiol. Pharmacol. 78 : 1324- 1329. On a determint le rCtablissement de propriCtCs mCtaboliques, n~orphologiqueset mkcaniques sp5cifiques d'unitts motrices rapides du muscle jambier anttrieur de rat aprks une dknervation partielle (n = 7), ou aprks une dCnervation partielle et une hknaisection de la moelle (n = 5 ) , et 19avonscompare au rCbblissement d'unitks tCmoins de rats du mCme 2ge (n = 7). Aprks 1 - 12 mois de retablissement, on a mesurd Ies proprietts mecaniques de chaque unite et appauvri Bes fibres en glycogene, en utilisant des techniques standard de stimulation de filaments de la racine ventrale. On a ueilisC des mkthodes hjistochimiques quantitatives pour determiner la section transverse et Les activites de succinate-dCshydrogCnase et d'a-glyc6ralphosphate dCshydrogCnase dans des fibres individuelles. La dknervation partielle a augment6 la surface moyenne des fibres, mais a diminuC l'activitk d'a-glyc6rolphosphate deshydrogtnase. La succinate dCshydrogCnase des groupes dknervks n'a pas variC. La variabilite dans les activitks enzymatiques et superficielles interfibres est demeuree inchangee. Toutefois. la relation entre les enzymes a Ctt aleCree par les deux modes de dinervation. L9activitC de sucsinate dkshydrogtnase fibrillaire a Ctk relliCe directement a la rCsistance h la fatigue et inversement & la tension tetanique. Ces rksultats suggkrent qu'une unit6 motrice ricupkre plusieurs de ses proprittCs malgrt des variations rnarqukes de la composition de 1'unitC provoqukes par la dknervation partielle. Be plus, une rkduction de I'activitt neuromusculaire des unites durant Ia rtorganisation a eu urn effet limit6 sur le retablissement. Mobs c l b : unite motrice, succinate dCshydrogCnase, or-glycCrolphosphate dCshydrogCnase, hdmisection de la rnoele. [Traduit par la rkdaction]
Introduction Following the partial denervation of skeletal muscle, the remaining intact motor units have the ability to enlarge their peripheral fields and compensate for the lost motoneurons (Brown et a&.1981; Brown and Hronton 1987; Kugelberg et a&. 1970; Luff et al. 1988; Luff and Torkko 1990; Tissenbaum and Parry 1991). This compensatory mechanism may be limited in that there appears to be a maximal capacity for neuronal sprouting (Brown et a&. 1981 ; Brown and Ironton 1987; Luff et al 1988; Luff and Torkko 1990). It has been suggested that there is a relationship between sprouting capacity and the efficacy of the sprouts with developmental age (Jacob and Robbins 1990; Lswrie ek a%. 1998; Rosenheirner 1990). In addition, neural and muscular activity have been reported to have some (Connold and Vrbova 1990, 1991; Ribchester and Taxt 1984) or no (Gardiner and Faltus 1986) impact on sprouting capacity. Partial denervation provides a rigorous test of the ability of a motor unit to recover its properties since the complement of fibres comprising a unit is altered. Despite the considerable [Author for correspondence, at the Department of Physical Therapy. Prlntcd
In
Canada / lmpr~mCau Canada
amount of information available concerning a muscle's response to partial denervation, the ability of individual units to reestablish their mechanical, morphological, and metabolic properties remains undefined. In addition, the role of neurombascular activity in the recovery from partial denervation is unclear. Therefore, this study was undertaken to determine the ability of rat tibidis anterior units to recover their mechanical and metabolic properties following partial denervation alone or in conjunction with hemispinal cord transection induced paralysis. Our observations suggest that the recovery of rat tibialis anterior fast units following partial denenation is rather robust. Furthermore, the reduction in neuromuscular activity (i .e. , hemispinalization) had only a limited impact on recovery.
Materials and methods Punial denemtion and spinal herntsectkosa All animal experimentation was conducted according to Canadian and University of Alberta guidelines for animal research. Twelve female Sprague - Dawley rats (220 -260 g body weight) were anesthetized (i.p., sodium pentobarbitone, 35 mg/kg) and the right ventral roots L3, U,and L5 were exposed under sterile conditions. To achieve varying degrees of partial denervation (PD group) of the tibialis anterior
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(TA), either one or two of the roots were cut and a portion of the distal aspect of the root(s) was excised. A hemisection of the spinal cord at T12 also was performed in five rats (HPD group). Subsequently, the animals were allowed to recover for 1 - 12 months. Seven additional age-rnatched control rats (no partial denervation or hemisection) were maintained under similar conditions.
irurtninul experirnentation The water given to each rat was replaced with a 5 % glucose solution to augment the glycogen levels of the TA 48 h prior to terminal experimentation. The rats were prepared for the isolation of single ventral root filaments under anesthesia using standard techniques (Gillespie et al. 1987). Briefly, a laminectomy was performed to expose the spinal cord from T13 to L6. The sciatic nerve and TA from both sides were dissected and all other hindlimb and hip muscles denervated. An EMG electrode was sewn into the fascia of the TA and a pair of fine wires were embedded in the musculature immediately surrounding the sciatic nerve, allowing indirect stimulation of the TA. The rat was mounted in a rigid frame with the legs held stationary by clamps. The distal tendon was attached to a force transducer with the skin surrounding the leg and spinal cord used to create mineral oil pools. A heating pad and radiant heat were used to maintain core and muscle temperature at 35 -37°C throughout the experiment. Determinatiora qf degree qf parria/ denemation The twitch and tetanic tensions of the right and left TA were determined by stimulation of the respective sciatic nerves. Subsequently, the twitch and tetanic tensions recorded from the stimulation of ventral roots L3, L4,or L5 were determined. If any force was measured in the previously cut right-side root, this was taken as evidence for regeneration of that root and the data from that animal were not included in the present data. A comparison of the force contributions between the right and left sides of the respective roots (the sums of L3, L4, and L5 of the left versus right multiplied by 100) provided ran estimation s f the degree of partial denervation (%PD) of the right TA. Single motor unit isolation and g!ycogen depletion A single unit was isolated using standard ventral root filament splitting techniques (Gillespie et a / . 1987). This unit was analyzed for tetanic force (200-ms trains with a 18-ms interpulse interval), fatigue resistance (188 pulses/s in 58-ms trains repeated each second for 2 min, with the ratio of the tension at 2 min relative to the initial tension providing a fatigue index), and sag during an unfused tetanus (800-n~strains with an interpulse interval 1.25 times the twitch contraction time). These mechanical properties were used to classify the units according to the criteria of Burke eF al. (1973) as fast fatigable, fast fatigue intermediate, or fast fatigue resistant. Subsequently, the unit was stimulated using intermittent trains of 5 pulses with a 10-ms pulse interval to deplete the fibres of glycogen. Tension was recorded throughout the stimulation and, if the decline in tension reached a steady state, the train rate was increased until tension continued to decline. Stimulation was maintained until the tension failed to recover following a brief rest period or until the stimulation response was lost. Muscle preparation and quantitative histoehemistry Following the glycogen depletion procedures, the TA was removed, weighed, and cut into blocks, which were mounted on cork and frozen in isopentane cooled in liquid nitrogen. Tissue sections (20 prn thick) were cut transversely from each block and prepared for the determination of glycogen using a periodic acid - Schiff's reaction (PAS). The tissue area that yielded the region with the greatest density of unit fibres depleted of glycogen was further analyzed using quantitative histochemical techniques described in detail elsewhere (Martin et al. 19880, l988b). Briefly, the PAS section was digitized using a computer-assisted image analysis system (PSICOM 232, Perceptive Systems. Inc., League City, Tex.). The optical densities (ODs) of a random sample of glycogen-depleted fibres (mean n = 17, range I10 - 33) were compared with the densities of fibres that did not appear to be depleted of glycogen (mean n = 32, range 20-48). 1%the distribution of ODs was not overlapping, those fibres with low
TABLE1. Summary of partial denervation and recovery procedures Animal
Roots cut for partial denervation
Root location of depleted unit
PD recovery (days)
Control I 2 3 4 5
6 7 PD 1 2 3 4 5 6 7 HPD 1 2 3 4 5
OD were considered to be part of the isolated muscle unit, while the remaining fibres (high OD) were taken to be representative of nonunit fibres. The failure of this analysis to generate two distinct distributions of different OD resulted in the exclusion of the muscle from further analysis. Some units with mechanical properties consistent with the slow fatigue resistant type were identified in the TA. However, none were successfully glycogen depleted based on the OD distribution in the PAS-prepared tissue sections. Therefore, the present findings are restricted to the fast unit populations of the rat TA. As a result. the nonunit fibres analyzed were also restricted to those that demonstrated a dark staining intensity (presumably from fast units) in the tissue section prepared for the qualitative assessment of myofibrillar ATBase (preincubation pH 8.8). In the muscles that contained a successfully isolated unit, serial tissue sections were cut and prepared for the determination of succinate dehydrogenase activity (SDH) (Blanco eb al. 1988), a-glycerolphssphate dehydrogenase activity (aGPD)(Martin et a / . 1988a), and a qualitative index of mysfibrillar ATPase staining intensity under alkaline (pH 8.8) preincubation conditions (Nwoye et a / . 1982). The same fibres were readily identified in the serial sections using the template of unit and nonunit fibres defined in the PAS section.
Data analysis Standard descriptive statistical procedures and linear regression analyses using Bearson's correlation coefficient were conducted using the PC-based statistical software program STATGRAPHICS (STSC, Inc., Rockville, Md.). Differences in dependent variables between groups and muscle units were analyzed using a two-way analysis of variance (ANOVA) and significant main effects and (or) interactions were analyzed using a Newman-Keuls multiple comparison procedure. Results were considered statistically significant if p < 0.05.
Results All successfully glycogen depleted units were classified according to their mechanical properties as fast fatigable, fast fatigue resistant, or fast fatigue intermediate. The sectioning of roots L3, L4, or L5 singularly, or in combination, yielded 2 - 80% partial denervation (Tables 1 and 2). Similar procedures in the presence of spinal cord hemisection resulted in
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TABLE2. Properties of the tibialis anterior motor units
Condition
Tetanic Fatigue tension %PD index (mN)
SDH (OD x 103/min), mean SD
Fibre area (p2). mean f SD Unit (CV)
+
Nonunit (CV)
Unit (CV)
Nonunit (CV)
GPD (OD x IO"/min), mean SD
+
Unit (CV)
Nonunit (CV)
Control
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1
2 3 4 5 6 7 PD 1 2 3 4 5 6 7
HPD 1 2 3 4 5
8-98% partial denervation. It should be noted that partial denervation above approximately 75 % resulted in the incomplete recovery of whole muscle force, suggesting a limit of sprouting potential in the rat TA. Table 2 provides the tetanic-tension and fatigue-resistance properties of the units; fibre area; and SBH (see d s o Fig. I) and GPD activity for the unit and nonunit fibres. There was a significant main effect for fibre area that resulted in a significant difference in the coefficient of variation (CV) between the motor unit and non-motor-unit fibers. A significant interaction effect revealed that the CV for fibre area was significantly different between motor unit and non-motor-unit fibres for both the control and partial denervated (PD) group but not the hemisected (HPD) group. Also, the CV for fibre area in the control nsnmotor unit was significantly different from the HPD nonmstor unit. There was a significant main effect for the CV of SBH and GPD activity. Thus, the CV of SDH and GPD activity was lower in the motor unit compared with the nonmotor units ( p < 0.05). No sther statistical differences were observed. The activities of SDH and GPD were found to be inversely related within the fibres of the control motor units (r = 0.$6, p < 0.05; Fig. 2). This relationship was absent in both the PD and HPD groups. Because SDH activity was unchanged Crelative to the control) in these latter groups, the decrease in GPD in the PD group and the elevated GPD in the HPD group may account for the differences. Mean SDH activity was positively related to fatigue resistance (Fig. 3). However, the relationship between fatigue resistance and SDH activity was only significant in the HPD units ( r = 0.98, p < 0.05). Finally, SDH activity was inversely related to tetanic tension but only achieved statistical significance in the control motor units (r = 0.78, p < 0.05; Fig. 4). One motor unit in the PD group displayed an inordinately high tetanic tension and SDH activity that could not be explained and was excluded from the analy-
sis. In general, the oppsiste relationship existed between the mechanical properties and GPD activity by virtue of the inverse relationship between GPD and SHD activities within the motor units.
It has been estabIished that in response to partial denervation the intact motoneurons enlarge their peripheral fields through collateral sprouting and reinnervate the denervated fibres (Brown et al. 1981; Brown and Ironton 1987; Kugelberg et a!. 1970; Luff et &a[. 1988; Luff and Torkko 1990; Tissenbaum and Parry 1991). Consequently, following sprouting, motor units are composed of fibres that previously belonged to disparate units with potentially very different properties. As a result, partial denervation provides an opportunity to investigate the strength sf the interrelationships among the properties of a unit. Previously, we reported that a considerable degree of variability in fibre size and metabolic enzyme activities existed within units of the cat soleus (Martin et al. 1988w) and tibilais anterior (Martin et a!. 19886). Although these findings are corroborated by the present data, as well as by other studies (Edstrom and KugeBberg 1968; Enad et a / . 1989), they are contrary to data that suggest that a unit may be composed of fibres with essentially identical properties (Nerneth et wl. 1981, 1986). Despite differences in the literature as te, the degree of the variability among the fibres of a unit, it is clear that the variance within a unit is significantly less than that found across a sample of different units in the same muscle. Therefore, we affirm our interpretation of these data to suggest that the properties s f a motor unit are regulated at the level of the "unit type" rather than at the level defined by a unit with fibres having identical properties (for further discussion see Martin et al. 1988a, 1988h). Some of the PI3 motonebarons enlarged their peripheral
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BELL ET AL.
Control Unit
PD Unit
HPD Unit
Control nonunit
P D nonunit
HPD wonunit
SDH
(OD/min, ~ 1 0 0 0 )
SDH. (8D/min, XI 000)
SBH
(BD/min, xdOOQ)
FIG.1. Frequency distributions of SDH activity for motor unit (open bars) and non-motor unit (solid bars) fibres from control (No. l), PD (No. $1, and HPD (No. 3).
fields by as much as 4-fold despite no change in the variability of fibre size or SDH and GPD activity within a unit. %tis likely that at least some of the fibres analyzed in these units originated from different units prior to the partial denervation. This
suggests that the factor(§) responsible for the regulation of unit properties is particularly puissant. It has been reported that motor unit fibres, after only 7 days of partial denervation, appeared to be more variable in size and SDH histochemical
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CAN. J . PHYSIOL. PHARMACQBL. VQL. 70, 1992
6 0
326 484 SDH Activity (OD x 10 )
1 60
640
800
FEG.2. The relationship between the mean activities of SDH and GPD from control (9, continuous rule), PD (u,dotted rule), and HPB (n, broken line) units. The enzymatic activities were inversely related in the control ( r = -0.05, ns) and HPB ( r = -0.39, ns) units.
SDH Activity (08 x 8 $1
FIG. 3. The relationship between mean SDH activity and fatigue index of the units. These properties were linearly related in the control (B. = 0.56, ns), PD (B. = 8.62, ns), and HPD(r = 0.98, p < 0.05) units. Symbols are as defined in Fig. 2.
staining intensity relative to controls (Gardiner et al. 1987). Therefore, fibres in partially denervated units may proceed through a phase of reorganization that alters the properties of the new fibres to those defined by the confines of the original composition of the unit. It is tempting to conclude that the innervating rnotoneuron may be responsible for the regulation of the fibre properties within the unit. However, motoneuronal control may not act independently since only SDH activity in the HPD units appeared to be regulated comparably with the control and PD units. The variability in fibre size and GPD activity across nonunit fibres in the HPD muscles was essentially equal to that
FIG. 4. The relationship between mean SDH activity and tetanic force of the units. These properties were inversely related in the control (r = -0.78, p < 0.05) and HPD (r = -0.73, ns) units. With the exception s f one unit in the PD group, a similar relationship existed (P = -0.47, ns). The regression line for the P B group is drawn with this unit excluded (in parentheses). Symbols are as defined in Fig. 2.
within a single unit, suggesting that muscular activity and load may be important regulatory factors. A similar conelusion has been drawn from the study of partial denervation in the presence of bungarotsxin-induced paralysis (Csnnold and Vrbova 1990, 1991). Furthermore, the reduced variability in fibre area and CPD activity across nonunit fibres in the HPD group (but not within a unit) could also be related to a shift in the entire motor unit population toward a more homogeneous population. This has been suggested to occur in the cat soleus following spinal cord transection (Cope et ak. 1986; Martin et al. 1 9 8 8 ~ ) . The lack sf an inverse relationship between SDM and GPD activities in the PD and HPD units appeared to be primarily a result of changes in GPD activity rather than SDH. Whether the change in CPD activity was secondary to the respective perturbations or reflects a lack of complete recovery by the motor units was unclear. The former possibility would be consistent with reports of an overall increase in GPD activity following spinal cord transection in cat soleus units (Martin et al. 1 9 8 8 ~ and ) medial gastrocnemius fibres (Jiang et a!. 1990). The general maintenance of the interrelationships among SDH activity, tetanic tension, and fatigue resistance in the PD and HPD units illustrates another level of control over the properties of a unit. Tetanic tensions increased in both groups of partially denervated motor units, while SDM activity remained similar to control levels, suggesting that the largest units prior to sprouting (with lower SDM activities) remained the largest after sprouting. The range in fatigue resistance across rat plantaris muscle units has been reported to decrease following partial denervation (Gardiner and Faltus 1986). These authors suggested that this change was due to the loss of units characterized by the two extremes of fatigability. The present study could not address this point owing to the lack of data from units classifiable as slow fatigue resistance. However, it is clear that partial denervation alone or in the presence
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BELL ET AL.
of hemispinal transection did not alter the fatigue resistance (or SDH activity) of the units. In summary, our data suggest that mstor units are capable of rather precise recovery s f their properties in the face of partial denervation (up to approximately 75 %). Consequently, a dynamic range of whole muscle function should remain following the recovery period. The recovery of some properties is compromised when muscular activity and load are reduced during the restoration period. However. the impact of this added perturbation was of limited consequence to the functional indices of recovery.
This work was funded in part by the Rick Hansen Man in Motion Legacy Fund and the Alberta Heritage Foundation for Medical Research. The technical assistance of Neil Tyreman was greatly appreciated. BIanco, C. E., Sieck, G . C.. and Edgerton, V. R. 1988. Quantitative histochemical determination of succinic dehydrogenase activity in skeletal muscle fibres. Histochem. J. 28: 230 -243. Brown, M. C., and Ironton, R. 1987. Sprouting and regression of neuromuscular synapses in partially denervated mammalian muscles. J . Physiol . (London), 278: 325 - 348. Brown, M. C., Holland, R. L., and Hopkins, W. G. 1981. Motor nerve sprouting. Annu. Rev. Neurosci. 4: 17 -42. Burke, R. E., Levine, D. N., Rsairis, P., and Zajac, F. E. 1973. Physiological types and histochemical profiles in motor units of cat gastrocnemius. 9. Physiol. (London), 234: 732 - 748. Connold, A. L., and Vrbova, G. 1990. The effect of muscle activity on motor unit size in partially denervated rat soleus muscles. Neuroscience, 34: 525 -532. Connold, A. L., and Vrbova, G. 1991. Temporary loss of activity prevents the increase of motor unit size in partially denervated rat soleus muscles. J. Physiol. (London), 434: 107- 119. Cope, T. C., Bodine, S. C . , Fournier, M.. and Edgerton, V. R. 1986. Soleus motor units in chronic spinal transected cats: physiological and morphological alterations. J. Neurophysiol. 55: 1202 1220. Edstrom, L., and Kugelberg, E. 1968. Histochemical composition, distribution sf fibers, and fatigability of single motor units. J. Neurol. Neurosurg. Psychiatry, 31: 424 -433. Enad. J. G., Fournier, M., and Sieck, G. C. 1989. Oxidative capacity and capillary density of diaphragm motor units. J. Appl. Physiol. 67: 620 - 627. Gardiner. P. F., and Faltus, R. E. 1986. Contractile responses of rat plantaris muscles following partial denervation, and the influence of daily exercise. Pfluegers Arch. 406: 51 -56. Gardiner, P., Michel, R., Olha, A., and Pettigrew, F. 1987. Force
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and fatiguability of sprouting motor units in partially denervated rat plantaris. Exp. Brain Res. 66: 597-606. Gillespie, J. M., Gordon, T., and Murphy, P. R. 1987. Motor units and histochemistry in rat lateral gastrocnemius and ssleus muscles: evidence for dissociation of physiological and histochemical properties after reinnervation. J . Neurophysiol. 57: 92 1-937. Jacob, J. M., and Robbins, N. 1990. Differential effects of age on transmission in partially denervated mouse muscle. J . Neurosci . 10: 1522- 1529. Jiang. B., Roy, R. R., and Edgerton, V. R. 1990. Enzymatic plasticity of medial gastrocnemius fibers in the adult chronic spinal cat. Am. J. Physiol. 259: 507 - 5 14. Kugelberg, E., Edstrom, L., and Abbruzzese, M. 1970. Mapping of motor units in experimentally reinnervated rat muscle. J. Neurol. Neurosurg. Psychiatry, 33: 3 19 -329. Lowrie, M. B., Shahani, U . , and Vrbova, G. 19%. Impairment of developing fast muscles after nerve injury in the rat depends upon the period of denervation. J. Neurol. Sci. 99: 249-258. Luff, A. R.. and Torkko, K. 1990. Long-term persistence of enlarged motor units in partially denervated hindlirnb muscles of cats. J . Neurophysiol. 64: 1261- 1269. Luff, A. R., Hatcher, D. D., and Toskko, K. 1988. Enlarged motor units resulting from partial denervation of cat hindlimb muscles. J. Neurophysiol. 59: 1377 - 1399. Martin, T. P., Bodine-Fowler, S. C., and Edgerton, V. R. 1 9 8 8 ~ . Coordination of electromechanical and metabolic properties of cat soleus mstor units. Am. J. Physiol. 255: 684 -693. Martin. T. P., Bodine-Fowler, S. C., Roy, R. R., Eldred. E. E., and Edgerton, V. R. 1988b. Metabolic and fiber size properties of cat tibialis anterior motor units. Am. J. Physiol. 255: 43 -50. Nemeth, P. M., Pette, D., and Vrbova, G. 1981. Comparison of enzyme activities among single muscle fibres within defined motor units. J. Physiol. (London), 311: 489 -495. Nemeth, P. M., Solanki, L., Gordon, D. A., Hamm, T. M., Reinking, R. M., and Stuart, D. G. 1986. Uniformity of metabolic enzymes within individual motor units. J. Neurosci. 6: $92 - 898. Nwoye. L., Mommaerts, W. F. H. M., Simpson, D. R., Serayderian, K., and Marusich, M. 1982. Evidence for a direct action of thyroid hormone in specifying muscle properties. Am. J. Physiol. 242: 401 -408. Ribchester, R. R., and Taxt, T. 1984. Repression of inactive motor nerve terminals in a partially denervated rat muscle after regeneration of active motor axons. J. Physiol. (London), 347: 497 - 5 11. Rosenheirner, J. L. 1990. Ultraterminal sprouting in innervated and partially denervated adult and aged rat muscle. Neuroscience. 38: 763 - 770. Tissenbaum, H. A., and Parry, D. J. 1991. The effect of partial denervation of tibialis anterior (TA) muscle on the number and size of motoneurons in TA motornaacleus of normal and dystrophic (C57BL dy2-'ldy2J) mice. Can. J. Physiol. Pharmacol. 69: 17691773.
