Hindlimb motor neurons require Cu/Zn superoxide dismutase for maintenance of neuromuscular junctions.

The role of oxidative damage in neurodegenerative disease was investigated in mice lacking cytoplasmic Cu/Zn superoxide dismutase (SOD), created by deletion of the SOD1 gene (SOD1(-/-)). SOD1(-/-) mice developed a chronic peripheral hindlimb axonopathy. Mild denervation of muscle was detected at 2 months, and behavioral and physiological motor deficits were present at 5-7 months of age. Ventral root axons were shrunken but were normal in number. The somatosensory system in SOD1(-/-) mice was mildly affected. SOD1(-/-) mice expressing Cu/Zn SOD only in brain and spinal cord were generated using transgenic mice expressing mouse SOD1 driven by the neuron-specific synapsin promoter. Neuron-specific expression of Cu/Zn SOD in SOD1(-/-) mice rescued motor neurons from the neuropathy. Therefore, Cu/Zn SOD is not required for normal motor neuron survival, but is necessary for the maintenance of normal neuromuscular junctions by hindlimb motor neurons.

4-Aminopyridine enhances motor evoked potentials following graded spinal cord compression injury in rats.

Although several experimental and clinical studies have demonstrated the ability of 4-aminopyridine (4-AP) to restore electrophysiological and/or behavioral function following chronic spinal cord injury, the mechanism by which this occurs remains unclear. Demonstration of efficacy in rat spinal cord injury has not been reported, evidently because even relatively mild spinal cord contusions that produce only minor permanent locomotor disturbances abolish hind limb myoelectric motor evoked potentials (mMEPs). In this study, mMEPs were recorded acutely 25 days following graded thoracic spinal cord compression in rats. mMEP amplitudes were significantly enhanced by a single, 2 mg/kg i.v. dose of 4-AP. mMEPs were increased in all rats showing some evoked responses initially, and also in some animals which had no responses prior to treatment. 4-AP was further found to increase the maximum following frequency of mMEPs in both normal and injured rats from about 0.1 Hz to between 1 and 10 Hz. These data suggest that 4-AP might act by enhancing synaptic efficacy, as well as enhancing conduction in spinal axons whose myelination has been rendered dysfunctional by trauma.

Insulin-like growth factor-I prevents development of a vincristine neuropathy in mice.

Vincristine is a commonly used antitumor agent whose major dose-limiting side-effect is a mixed sensorimotor neuropathy. To assess whether insulin-like growth factor-I (IGF-I), a neurotrophic agent that supports the survival of motoneurons and enhances regeneration of motor and sensory neurons, could prevent the peripheral neuropathy produced by vincristine, mice were treated with both vincristine (1.7 mg/kg, i.p., 2 x /week) and/or IGF-I (0.3 or 1 mg/kg, s.c. daily) for 10 weeks. In mice treated with vincristine alone, there was evidence of a mixed sensorimotor neuropathy as indicated by changes in behavior, nerve conduction and histology. Caudal nerve conduction velocity was significantly slower in mice treated with vincristine alone as compared with vehicle-treated mice. Vincristine treatment alone also significantly increased hot-plate latencies and reduced gait support and stride length, but not toe spread distances. The effects of vincristine were accompanied by degeneration of sciatic nerve fibers and demyelination, indicating a peripheral neuropathy. IGF-I (1 mg/kg, s.c.) administered to vincristine-treated mice prevented the neurotoxic effects of vincristine as measured by nerve conduction, gait, response to noxious stimuli and nerve histology. At a lower dose of 0.3 mg/kg administered s.c., IGF-I partially ameliorated the neuropathy induced by vincristine as this dose only prevented the change in nerve conduction and hot-plate latencies. IGF-I administered alone had no effect on any of these parameters. These results suggest that IGF-I prevents both motor and sensory components of vincristine neuropathy and may be useful clinically in preventing the neuropathy induced by vincristine treatment.

Histological and functional evaluation of experimental spinal cord injury: evidence of a stepwise response to graded compression.

Most experimental spinal cord injury studies described to date have relied on a limited number of injury gradations, and have tacitly assumed that outcome (functional, histological, and/or neurophysiological) is a monotonically graded function of injury severity. In contrast, the present study provides evidence that functional and morphological outcome after spinal cord compression injury may occur in a discontinuous, non-graded manner in response to linearly graded injury levels. The thoracic spinal cord of adult rats was transiently compressed to thicknesses from 1.8 to 0.8 mm in 0.2 mm steps, or sham injury was administered. Open field motor behavior and segmental reflexes were evaluated up to 21 days post injury and correlated with histological measures and injury level. The highest correlation was between histological outcome and open field motor scores. Among the six injury groups, only three significantly different outcomes were apparent in the open field, reflex, and histological measures, consisting of the injury group pairs 1.8/1.6, 1.4/1.2, and 1.0/0.8 mm. At day 21, the 1.8/1.6 mm injury groups were also indistinguishable from the sham injury group. The implications of these findings in terms of therapeutic studies are discussed. Comparison of the temporal outcome patterns among contusion and compression injuries in rats and other species also revealed a significant species difference: a period of delayed or secondary functional loss reported in the guinea pig was not present in the rat.

MASCIS evaluation of open field locomotor scores: effects of experience and teamwork on reliability. Multicenter Animal Spinal Cord Injury Study.

The Multicenter Animal Spinal Cord Injury Study (MASCIS) adopted a modified 21-point open field locomotor scale developed by Basso, Beattie, and Bresnahan (BBB) at Ohio State University (OSU) to measure motor recovery in spinal-injured rats. BBB scores categorize combinations of rat hindlimb movements, trunk position and stability, stepping, coordination, paw placement, toe clearance, and tail position, representing sequential recovery stages that rats attain after spinal cord injury. A total of 22 observers from 8 participating centers assessed 18 hindlimbs of 9 rats at 2-6 weeks after graded spinal cord injury. The observers were segregated into 10 teams. The teams were grouped into 3 cohorts (A, B, and C), consisting of one experienced team from OSU and two non-OSU teams. The cohorts evaluated the rats in three concurrent and sequential sessions. After viewing a rat for 4 min, individual observers first assigned scores without discussion. Members of each team then discussed and assigned a team score. Experience (OSU vs. non-OSU) and teamwork (individual vs. team) had no significant effect on mean scores although the mean scores of one cohort differed significantly from the others (p = 0.0002, ANOVA). However, experience and teamwork significantly influenced reliability of scoring. OSU team scores had a mean standard deviation or discordance of 0.59 points, significantly less than 1.31 points for non-OSU team scores (p = 0.003, ANOVA) and 1.30 points for non-OSU individual scores (p = 0.001, ANOVA). Discordances were greater at the upper and lower ends of the scale, exceeding 2.0 in the lower (< 5) and upper (> 15) ends of the scale but were < 1.0 for scores between 4 and 16. Comparisons of non-OSU and OSU team scores indicated a high reliability coefficient of 0.892 and a correlation index (r2) of 0.894. These results indicate that inexperienced observers can learn quickly to assign consistent BBB scores that approach those given by experienced teams, that the scores are most consistent between 4 and 16, and that experience improves consistency of team scores.

Myoelectric evoked potentials versus locomotor recovery in chronic spinal cord injured rats.

The purpose of this study was to determine the utility of descending evoked potentials in evaluating functional recovery in rats after spinal cord contusion injury. Rats received thoracic contusions at T9 using a controlled-displacement impactor. They were evaluated for 5 weeks postinjury using auditory startle responses (ASR) while alert, or by cerebellar motor evoked potentials (CMEP) while anesthetized. ASR and CMEP were recorded electromyographically from forelimb and hindlimb muscles. Open field locomotor performance was also assessed and recovered to almost normal levels by 3 weeks postinjury. Histologic analysis of the injury site indicated that the contusions destroyed approximately 70% of the cross-sectional area of the cord. Although the remaining 30% was sufficient to preserve nearly normal locomotor behavior, ASR and CMEP amplitudes in hindlimb flexors and extensors were reduced by 90% or more after injury and showed virtually no recovery. Significant ASR and CMEP responses were present in the cutaneous trunk muscles of the lower torso after injury. These muscles are innervated via peripheral nerves originating at cord levels above the injury. Multi-wave field potentials normally recorded from the dorsal cord surface in response to cerebellar stimulation were absent in injured rats, suggesting minimal if any activation of segmental neurons via the pathways normally mediating CMEP. The tracts mediating ASR and CMEP thus appear to be highly sensitive to mild spinal cord trauma but are evidently not essential for support or walking.

Regulation by synapsin I and Ca(2+)-calmodulin-dependent protein kinase II of the transmitter release in squid giant synapse.

1. Presynaptic or simultaneous pre- and postsynaptic voltage-clamp protocols were implemented in the squid giant synapse in order to determine the magnitude and time course of the presynaptic calcium current (ICa) and its relation to transmitter release before and after presynaptic injection of proteins. These included several forms of synapsin I, calcium-calmodulin-dependent protein kinase II (CaM kinase II) and avidin. 2. The quantities and location of these proteins were monitored by fluorescence video-enhanced microscopy during the electrophysiological measurements. 3. Presynaptic injection of dephosphorylated synapsin I inhibited synaptic transmission with a time course consistent with diffusion of the protein through the terminal and action at the active release zone. A mathematical model relating the diffusion of synapsin I into the terminal with transmitter release was developed to aid in the interpretation of these results. 4. Synapsin I inhibition of transmitter release was reversible. 5. The action of synapsin I was highly specific, as phosphorylation of the tail region only or head and tail regions prevented synapsin I from inhibiting release. 6. Injections of heat-treated synapsin I or of avidin, a protein with a size and isoelectric point similar to those of synapsin I, had no effect on transmitter release. 7. CaM kinase II injected presynaptically was found to facilitate transmitter release. This facilitation, which could be as large as 700% of the control response, was related to the level of penetration of the enzyme along the length of the preterminal A mathematical model of this facilitation indicates a reasonable fit between the distribution of CaM kinase II within the terminal and the degree of facilitation. 8. The overall shape of the postsynaptic response was not modified by either synapsin I or CaM kinase II injection. 9. The data suggest that, in addition to releasing transmitter, calcium also penetrates the presynaptic cytosol and activates CaM kinase II. When activated, CaM kinase II phosphorylates synapsin I, which reduces its binding to vesicles and/or cytoskeletal structures, enabling more vesicles to be released during a presynaptic depolarization. The amplitude of the postsynaptic response will then be both directly and indirectly regulated by depolarization induced Ca2+ influx. This model provides a molecular mechanism for synaptic potentiation.

Assessment of functional recovery after spinal cord injury in rats by reticulospinal-mediated motor evoked responses.

Auditory startle reflexes (ASR) and cortico-myoelectric evoked potentials (CMyEP) were investigated as possible tests of descending motor function in a rat spinal cord injury model. ASR, which consist of stereotyped myoelectric responses recorded in limb and axial muscles to brief loud tones, were found to provide a simple, objective, and reliable measure of motor recovery after spinal cord injury (SCI). While ASR are easily recorded in awake rats, they are blocked by anesthetics, and thus cannot be recorded during the acute injury period. ASR were compared with CMyEP, which can be recorded while the animal is anesthetized. CMyEP were found to produce large myoelectric responses in the vastus lateralis and tibialis anterior hindlimb muscles of the rat similar to ASR except that latencies were approximately 3 msec earlier. Both ASR and CMyEP tended to be bilaterally symmetric regardless of the stimulus configuration, and threshold for responses were the same for both muscles in both hindlimbs. The results suggest that CMyEP may be related to ASR and thus mediated partly by reticulospinal pathways. Evidence supporting this hypothesis is reviewed.

Phosphorylation-dependent inhibition by synapsin I of organelle movement in squid axoplasm.

Synapsin I, a neuron-specific, synaptic vesicle-associated phosphoprotein, is thought to play an important role in synaptic vesicle function. Recent microinjection studies have shown that synapsin I inhibits neurotransmitter release at the squid giant synapse and that the inhibitory effect is abolished by phosphorylation of the synapsin I molecule (Llinas et al., 1985). We have considered the possibility that synapsin I might modulate release by regulating the ability of synaptic vesicles to move to, or fuse with, the plasma membrane. Since it is not yet possible to examine these mechanisms in the intact nerve terminal, we have used video-enhanced microscopy to study synaptic vesicle mobility in axoplasm extruded from the squid giant axon. We report here that the dephosphorylated form of synapsin I inhibits organelle movement along microtubules within the interior of extruded axoplasm and that phosphorylation of synapsin I on sites 2 and 3 by calcium/calmodulin-dependent protein kinase II removes this inhibitory effect. Phosphorylation of synapsin I on site 1 by the catalytic subunit of cAMP-dependent protein kinase only partially reduces the inhibitory effect. In contrast to the inhibition of movement along microtubules seen within the interior of the axoplasm, movement along isolated microtubules protruding from the edges of the axoplasm is unaffected by dephospho-synapsin I, despite the fact that the synapsin I concentration is higher there. Thus, synapsin I does not appear to inhibit the fast axonal transport mechanism itself. Rather, these results are consistent with the possibility that dephospho-synapsin I acts by a crosslinking mechanism involving some component(s) of the cytoskeleton, such as F-actin, to create a dense network that restricts organelle movement. The relevance of the present observations to regulation of neurotransmitter release is discussed.

Comparison of vestibular and auditory startle responses in the rat and cat.

Cats, humans, and many other animals show stereotyped EMG responses in limb and axial muscles if suddenly dropped into free-fall. In cats, these free-fall responses (FFR) consist of highly synchronized bursts in most limb and axial muscles at 18-22 ms. We have used FFR to evaluate descending motor function and recovery in chronic spinal injured cats. Here FFR are compared with auditory evoked startle reflexes (ASR) in the hindlimb muscles of the rat and cat to determine whether they are related, and whether they could be used to evaluate descending motor function in the rat. ASR and FFR in the two species were similar except that the earliest components for both responses occurred around 9 ms in the rat versus 18-20 ms in the cat. Also, FFR in cats were usually more consistent and greater in amplitude during repeated drops than in rats, while the converse was true for ASR. Rat FFR amplitudes increased significantly after administering ketamine or 4-aminopyridine (4-AP), especially with both drugs together, while ASR amplitudes did not. FFR in cats recorded under ketamine analgesia were not normally improved by 4-AP. Finally, both FFR and ASR were suppressed by pentobarbital, chloralose, or motor activity. These data suggest that: (1) FFR appears to be a vestibular evoked startle reflex; (2) in the rat, ASR should be useful as a test of descending motor function following spinal injury, and (3) the combination of ketamine and 4-AP may be useful in revealing the presence of functional spinal pathways after CNS trauma.

Nonlinear muscle recruitment during intramuscular and nerve stimulation.

Future progress in neuromuscular prostheses will depend on developing techniques for stimulating paralyzed muscle, especially utilizing neuromuscular stimulation. We have found nonlinear force versus stimulus amplitude characteristic (recruitment) curves in the gastrocnemius-soleus-plantaris muscle group of the cat in response to stimulation of the tibial nerve near the muscle entry point. Such response characteristics are undesirable in neuromuscular control systems. Nonlinear recruitment curves usually consisted of two regions in which force increased linearly with stimulus amplitude, separated by a "plateau" region in which force was relatively constant. The two linear regions were associated with activation of separate neuromuscular compartments (lateral or medial gastrocnemius, plantaris, soleus, or subdivisions of those muscles). When the stimulated myoelectric responses from these compartments were plotted versus stimulus amplitude, the region of recruitment between threshold and saturation often did not appreciably overlap for different compartments, suggesting that the axons innervating those compartments were physically segregated within the nerve from axons innervating other compartments. Correlation coefficients between force and stimulated myoelectric response were very high (up to R2 = 0.99) when using a composite curve produced by averaging myoelectric response curves recorded from each of the active compartments. By dividing the tibial nerve into its component bundles or fascicles and stimulating each in turn, it was possible to show that individual bundles innervate non-overlapping groups of muscle compartments, and that recruitment of the nerve bundles over different threshold ranges could account for the nonlinear force/stimulus response curves initially observed. The presence of separate innervation of muscles or compartments by fascicles should be an important factor in designing functional neuromuscular stimulation (FNS) systems.

Augmentation by 4-aminopyridine of vestibulospinal free fall responses in chronic spinal-injured cats.

This study examines the effect of the potassium channel blocker 4-aminopyridine (4-AP) on free fall responses (FFR) in the hindlimb muscles of chronically spinal injured cats. The thoracic spinal cord of 7 adult female cats was injured by a standardized contusion method. At 3-7 months post-injury the FFR in 6 hindlimb muscles was recorded electromyographically in each animal, under ketamine sedation. The normal short-latency response to a sudden drop was severely attenuated in all injured animals and practically undetectable in 2 cases. Within 15 min following intravenous administration of 1 mg/kg 4-AP, there was profound augmentation of the amplitude of the FFR and a tendency toward normalization of latency in all animals, though the normal amplitude range was not attained. The same 4-AP dose produced a relatively small increase of FFR amplitude in only 2 of 4 normal, uninjured animals tested. The data are consistent with previous observations that low doses of 4-AP restore conduction in some critically demyelinated axons, and provide support for the hypothesis that conduction block in surviving axons is responsible for a proportion of the dysfunction in chronic spinal injury. Augmentation of FFR in injured animals may also result partly from increased transmitter release in both spinal cord and periphery, due to the presynaptic effects of 4-AP.

Considerations in designing acceptable neuromuscular stimulation systems for restoring function in paralyzed limbs.

Recent progress in the field of functional neuromuscular stimulation (FNS), that is, the restoration of purposeful movement to paralyzed limbs via stimulation, has enabled paraplegic individuals to stand up, walk, and even climb stairs (with assistance) and quadriplegics to hold and manipulate utensils and useful objects. A number of experimental and clinical FNS systems are considered, emphasizing the algorithms used to regulate movement under a variety of conditions (isometric, isotonic, dynamic load, reciprocal control) and the physiologic problems encountered. Further development of FNS systems is required to achieve patient acceptability in daily living. Three important factors will be use of implanted intramuscular or nerve stimulating electrodes, quantitative documentation of FNS-produced movements, and incorporation of force, position, and other modes of feedback to both the controller and the patient.

The vestibulospinal free fall response: a test of descending function in spinal-injured cats.

A major problem in spinal cord injury research is quantification of motor function in animals. Most investigators in the field currently use neurologic scoring systems, relying on subjective observations of complex behaviours and assigning scores based on arbitrary criteria. These scoring scales are prone to observer bias and are nonspecific. We describe here a simple, reproducible, noninvasive, and objective test of a limited aspect of spinal motor function in cats, based on a well-known involuntary response of animals to sudden free fall. Free fall responses, or FFRs, have been studied in many species, including man, and are thought to be carried in ventral and lateral column pathways, i.e., vestibulospinal, reticulospinal, and rubrospinal tracts. We recorded the FFRs from hind and forelimb muscles of 100 cats before and after thoracic spinal cord injury. Hindlimb FFRs were shown to have three quantifiable components: a fast synchronous activation (E1) followed by a short silent period during which spinal segmental reflexes are inhibited (I1) and a late desynchronized excitatory burst (E2). Thoracic spinal injury produced hindlimb FFR losses ranging from greatly reduced amplitude to complete absence of response. Residual FFRs correlated with the extent of ventral column preservation and locomotory ability. Individual FFR components can be preserved. For example, some injured cats exhibited only 11 responses. Our work suggests that FFRs are a reliable and sensitive test of motor recovery in spinal cord injury.

A system for evaluation and exercise-conditioning of paralyzed leg muscles.

The purpose of this project was to develop instrumentation and protocols in which electrical stimulation is used to induce exercise in paralyzed quadriceps muscles strength and endurance evaluation and conditioning. A computer-controlled electrical stimulation system, using surface electrodes, automatically regulates the bouts of leg extension exercise. Load weights attached just above the ankles can be progressively increased over a number of training sessions in such a manner that a measure of the fitness of the legs can be obtained. With three exercise sessions per week for 9 weeks, the strength and endurance of the quadriceps muscles of two paraplegic and four quadriplegic subjects were gradually and safely increased. During exercise at a means load weight of 5.4 kg, means heart rate did not rise above rest, whereas systolic blood pressure increased about 20 mm Hg, and skin temperature above the active muscles increased about 1.75 degrees C. Such exercise conditioning appears to be safe and may provide important health benefits, including improved fitness of the muscles and bones, better circulation in the paralyzed limbs, and enhanced self-image. Conditioned electrically stimulated paralyzed leg muscles may be used for locomotion in conjunction with special vehicles.

Locomotion via paralyzed leg muscles: feasibility study for a leg-propelled vehicle.

Functional electrical stimulation has been used to restore some degree of controllable movement to paralyzed muscle. The purpose of this study was to demonstrate the feasibility of using electrically stimulated paralyzed leg muscles to propel a wheelchair-type vehicle. For this, a conventional manual wheelchair was modified by the addition of a drive system which permits forward propulsion by reciprocating movements of the legs. A battery-powered electrical stimulator using surface electrodes over the quadriceps muscles controls locomotive characteristics. This vehicle has been successfully operated by paraplegic and quadriplegic test subjects. Advantages of using paralyzed leg muscles for locomotion may include improvement in locomotive capability, circulation in the lower extremities, cardiovascular and respiratory fitness, strength and size of the exercised muscles and bones, and self-image.

Effects of arrested cerebellar development on locomotion in the rat. Cinematographic and electromyographic analysis.

The activity of the rat hindlimb during treadmill stepping was studied in normal adult rats and in rats in which cerebellar development was interfered with by early-postnatal focal X-irradiation. Based on cinematographic and electromyographic data from over 100 step cycles in 15 normal rats, correlations were made between joint angles and muscle activity to obtain a detailed picture of the locomotor pattern of the rat hindlimb. It was possible to relate most of the features of limb movement to activity in one or more of six primary flexors and extensors of the hindlimb. Compared with available data in the cat or dog, the joint angle curves were similar in shape except that the knee joint angle was usually greater at foot contact than at lift-off, while in cats and dogs the reverse is usually the case. This difference is due to a more crouched stepping posture in the rat in which the leg is not extended as far backward as in the cat or dog. It was also noticed that there was more side-to-side bowing of the spine in the rat during stepping. Finally, in rats there was no correlate to the stance phase burst seen in the semitendinosus in cats. In rats with cerebellar X-irradiation there was little effect on the stepping cycle if the animal's equilibrium was maintained externally, either by a supporting harness or by immersion in water (swimming). However, when stepping without external support, animals were unable to adequately compensate for perturbations in equilibrium, resulting in an ataxic gait. This problem was compound by the presence of high-frequency (18 Hz) tremor which, by producing hyper- or hypotonia during critical periods of stepping such as foot placement or during corrective reflex movements, was a major disturbing force to the animal's equilibrium.