Properties of spinal motoneurons and interneurons in the adult turtle: provisional classification by cluster analysis.

The purpose of the present study was to compare, in motoneurons (MNs) vs. interneurons (INs), selected passive, transitional, and active (firing) properties, as recorded in slices of lumbosacral spinal cord (SC) taken from the adult turtle. The cells were provisionally classified on the basis of (1) the presence (in selected INs) or absence (MNs and other INs) of spontaneous discharge, (2) a cluster analysis of selected properties of the nonspontaneously firing cells, (3) a comparison to previous data on turtle MNs and INs, and (4) a qualitative comparison of the results with those reported for other vertebrate species (lamprey, cat). The provisional nomenclature accommodated properties appropriate for solely MNs (Main MN group) vs. nonspontaneously firing INs (Main IN-N) vs. spontaneously firing INs (IN-S) and for neurons with two degrees of intermediacy between the Main MN and the Main IN-N groups (Overlap MN, Overlap MN/IN). Morphological reconstructions of additional cells, which had been injected with biocytin during the electrophysiological tests, were shown to provide clear-cut support for the provisional classification procedure. The values for the measured parameters in the 96 tested cells covered the spectrum reported previously across adult vertebrate species and were robust in measurements made on different SC slices up to 5 days after their removal from the host animal. The interspecies comparisons permitted the predictions that (1) our Main MN and Overlap MN cells would be analogous to two MN types that innervate fast-twitch and slow-twitch skeletomotor muscle fibers, respectively, in the cat, and (2) the MNs in our Overlap MN/IN group probably innervate slow (nontwitch, tonic) muscle fibers whose presence has recently been established in the turtle hindlimb. In summary, the results bring out the utility of the SC slice preparation of the turtle for study of spinal motor mechanisms in adult tetrapod vertebrates, particularly as an adjunct to the in vivo cat, because of the ease with which robust measurements can be made of the active properties of both MNs and INs.

Neurobiology of muscle fatigue.

Muscle fatigue encompasses a class of acute effects that impair motor performance. The mechanisms that can produce fatigue involve all elements of the motor system, from a failure of the formulation of the descending drive provided by suprasegmental centers to a reduction in the activity of the contractile proteins. We propose four themes that provide a basis for the systematic evaluation of the neural and neuromuscular fatigue mechanisms: 1) task dependency to identify the conditions that activate the various mechanisms; 2) force-fatigability relationship to explore the interaction between the mechanisms that results in a hyperbolic relationship between force and endurance time; 3) muscle wisdom to examine the association among a concurrent decline in force, relaxation rate, and motor neuron discharge that results in an optimization of force; and 4) sense of effort to determine the role of effort in the impairment of performance. On the basis of this perspective with an emphasis on neural mechanisms, we suggest a number of experiments to advance our understanding of the neurobiology of muscle fatigue.

Coexistence of twitch potentiation and tetanic force decline in rat hindlimb muscle.

An experimental protocol designed to assess fatigability in motor units has been applied to two hindlimb muscles of anesthetized adult rats to study the effects of whole-muscle fatigue on the isometric twitch. Both soleus and extensor digitorum longus exhibited a linear relationship between fatigability (i.e., force decline after a 360-s fatigue test) and the magnitude of the twitch force following the fatigue test. Twitch force after the fatigue test was potentiated (i.e., greater than the value before the fatigue test) in many muscles, despite the development of considerable fatigue. This coexistence of fatigue and twitch potentiation was observed in 7% (5/70) of soleus and 48% (31/64) of extensor digitorum longus muscles. The coexistence was exhibited only by the least fatigable muscles of the fast-contracting extensor digitorum longus. The extensor digitorum longus muscles that did not exhibit twitch potentiation probably experienced a higher proportion of muscle-fiber inactivation, such as due to failure of neuromuscular propagation, that was induced by the fatigue regimen.

Reduced motor unit activation of muscle spindles and tendon organs in the immobilized cat hindlimb.

Six weeks of limb immobilization of a healthy muscle (cat tibialis posterior) at a short length resulted in a significant reduction of mean fiber area for all fiber types (I, 71% of control; IIa, 77% of control; IIb, 79% of control), whereas fiber type proportions were unchanged. For motor units, there was a reduction in peak tetanic force (type slow > fast fatigue resistant > fast fatigable); an increase in the twitch-to-tetanus ratio for fast fatigue-resistant and slow units; and no effect on the twitch force, twitch time course, or fatigability. The reduction in peak force was greater than expected because of fiber atrophy in slow units. Immobilization had a minimal effect on muscle spindle afferent (Ia and spindle group II) responses to a ramp-and-hold stretch of the passive muscle. Tendon organ (Ib) afferents had an increased responsiveness to stretch after immobilization but only when the muscle was stretched from a short resting length. However, immobilization reduced the modulation of muscle afferent discharge in response to tetanic contractions of single motor units. The decline in responsiveness of spindles was a result of the reduced tetanic force of motor units. In contrast, tendon organs in immobilized muscle were twice as likely to convey no information on the contraction of a single motor unit and were more likely to be unloaded, suggesting that immobilization caused the functional denervation of some muscle fibers. Thus the responses of muscle spindles and tendon organs in immobilized muscle reflected atrophic changes in extrafusal fibers but did not provide evidence for substantial disturbance of receptor function.

Electrophysiological and morphological properties of neurons in the ventral horn of the turtle spinal cord.

In this report, we present recent findings on the electrophysiological and morphological properties of spinal motoneurons (MNs) and interneurons (INs) of the adult turtle which were studied in slices of the spinal cord. The range of values for the measured electrophysiological parameters in 96 tested cells included: resting potential, -57 to -83 mV; input resistance, 2.5-344 M omega; time constant, 2.5-63 ms; rheobase current, 0.04-5.3 nA; after-hyperpolarization (AHP) duration, 72-426 ms; AHP half-decay time; 11-212 ms; and, slope of the stimulus current-spike frequency relationship, 3.4-235 Hz/nA. For another 20 cells, we made both morphological and electrophysiological measurements (the latter values within the above ranges). Their ranges in morphological properties included: soma diameter, 20-54 microm; soma surface area, 299-2045 microm2; soma volume, 2.3-45 microm3 x 10(4); rostro-caudal dendritic projection distance, 150-1200 microm; and, sum of dendritic lengths, 1.5-16 microm x 10(3). The emphasized findings include: 1) the quality and robustness of the intracellular recordings, which enabled accurate measurement of the action potential's shape parameters (spike, afterhyperpolarization [AHP]); 2) the substantial AHP of the INs' AP; 3) no single action-potential shape parameter (nor combination of parameters) being cardinal for its (or their combined) changes matching the profile of the initial and later phases of spike-frequency adaptation; 4) the utility and flexibility of a cluster analysis (using varying combinations of passive, transitional and active cell properties) for providing a provisional classification of low (like cat S) and high (like cat F) threshold MNs, and groups of INs with non-spontaneous versus spontaneous discharge; 5) the clear-cut morphological confirmation of the provisional classification strategy; 6) the basis for testing the possibility that one of the provisionally classified MN types innervates non-twitch muscle fibers; and 7) the heuristic value of comparing the properties of MNs versus INs across vertebrate species, with an emphasis on the lamprey, turtle, and cat.

An effective sleeving technique in nerve repair.

This report describes the use of a porous polymeric sleeve (Gore-tex) to direct nerve fiber growth after axotomy. Select nerves of the triceps surae muscles in 5 adult cats were surgically isolated, sectioned, and crossed or self- reunited . A piece of Gore-tex, 15 mm in length, was compressed to 5 mm and sleeved over each distal nerve end. The appropriate proximal and distal ends were stitched together, and the Gore-tex stretched back to its original length over the suture junction. The effectiveness of the Gore-tex sleeve was assessed 4-15 months post-operatively. Electrophysiological measurements of muscle force and dorsal root volleys revealed a complete absence of unintended reinnervation and a regeneration that was more substantial for motor than sensory axons. Finally, serial histological cross-sections were prepared for each nerve above, below and at the cross union. There was no evidence of nerve tissue invading the Gore-tex wall.

Detection of synchrony in the discharge of a population of neurons. I. Development of a synchronization index.

A test for synchronization among the spike trains of muscle afferents or motor units is described which utilizes averages of neurograms and rectified neurograms. Synchronization is quantified by the increase of a synchronization index Is above a theoretical value for asynchrony. The dependence of the Is on signal amplitude and certain experimental conditions and a method of estimating confidence limits for the test are presented.

Detection of synchrony in the discharge of a population of neurons. II. Implementation and sensitivity of a synchronization index.

This report describes the use of a synchronization index (Is; Hamm et al., 1985a) and its sensitivity to various forms and degrees of synchrony between spike trains. The dependence of the Is on signal-to-noise ratio, the number of synchronized spike trains and their degree of synchrony is shown in analog and digital simulations. These simulations and a comparison with peristimulus time histograms under conditions of induced synchrony reveal that the Is is a sensitive measure of synchronization in a population of spike trains.

Measurement of axonal conduction velocity in single mammalian motor axons.

In deeply anesthetized cats, determinations of motor-axonal conduction velocity (CV) were made using extracellular potentials recorded from single, functionally isolated motor axons innervating the muscle tibialis posterior. Axons were activated by suprathreshold electrical stimulation at the ventral-root level. Action potentials were recorded with 3 bipolar electrodes located on the muscle nerve at the level of the popliteal fossa. The most proximal and distal of the bipolar muscle-nerve electrodes were 16.4-22.0 mm apart. Estimates were made of CV from ventral root to muscle nerve (conventional CV) and between the proximal and the distal pairs of muscle-nerve electrodes (muscle-nerve CV). An evaluation was based on comparison of these CVs, estimates of uncertainties in time and distance measurements and simulations of the effects of recording conditions on CV estimates. The analysis indicated that the uncertainty in the conventional CV measurement of mammalian motor axons is at least +/- 2%. However, variability may be as great as 20% between muscle-nerve CV measurements from different experiments, probably due to such factors as regional variation in CV and differences in recording configuration.

The motor units of cat medial gastrocnemius: speed-size relations and their significance for the recruitment order of mortor units.

The interrelationships between axonal conduction velocity, tetanic tension, twitch contraction time and rate of force development during a tetanus have been studied in 126 motor units from 12 cat medial gastrocnemius muscles. While the range of axonal conduction velocities for slow- (contraction time greater than 45 msec) and fast-twitch units overlapped, the mean conduction velocity for the slow-twitch group was significantly lower. No difference could be found between the axonal conduction velocities of the fast non-fatiguing and fast fatiguing units. Within individual experiments, few significant correlations were found between conduction velocity and tetanic tension or contraction time for the fast- and slow-twitch units. Some correlations did appear when data from these populations were pooled, but such results are shown to be misleading. Some weak correlations were found between motor unit contraction strength and twitch contraction time. The rate of rise of isometric force development was found to be most strongly related to tetanic tension and only weakly related to contraction time. The ordering of motor units according to contraction strength reveals the association of motor unit mechnical properties to be ideally suited for the dual role of medial gastrocnemius as a postural and powerful phasic muscle.

Stimulus-related correlations between medial gastrocnemius muscle tension and homonymous motoneuron membrane potential result from non-linearities.

In anesthetized cats, medial gastrocnemius motor units (MUs) were electrically stimulated via their ventral-root axons with independent random patterns. Muscle tension and homonymous alpha-motoneuron (alpha-MN) membrane potential fluctuations in response to these stimuli were recorded simultaneously. Cross-correlating these two signals in temporal relation to the stimuli showed the covariance of the two signals around their respective means. With moderate to strong MU-MN couplings indicated by clear and large average membrane potential changes (PSP trajectories), the correlation coefficients tended to be positive at times when the PSP hyperpolarized, and vice versa. These relations are probably caused by the non-linear behavior of MUs, muscle receptors and afferent pathways. Thus, the signal transfer from motor efferents back to homonymous alpha-MNs depended upon the level of background tension, upon which MU twitches were superimposed.

Contractions of single motor units are reflected in membrane potential changes of homonymous alpha-motoneurons.

In anesthetized cats 3 medial gastrocnemius (MG) motor units (MUs) were electrically activated with independent pseudorandom patterns at physiological mean rates. Recordings of isometric MG tension (T) and membrane potential changes (PSPs) in MG alpha-motoneurons (MNs) were averaged with respect to each stimulus train. The resulting T and PSP trajectories displayed the responses of each MN to the 3 average MU twitches. These responses could vary from no response to a sequence of depolarizing and hyperpolarizing waves in a PSP trajectory. The results are consistent with the known behavior of muscle stretch receptors to muscle and MU twitches and the receptors' connections to homonymous MNs.

Linear and non-linear summation of alpha-motoneuron potential changes elicited by contractions of homonymous motor units in cat medial gastrocnemius.

An analysis is made of linear and non-linear summation of the motor unit (MU) tension and motoneuron (MN) membrane potential (PSP) trajectories evoked by stimulation of MUs as previously reported. In most cases, summation of both averaged responses was linear. Seldom, PSPs summed non-linearly, then usually coinciding with non-linear summation of the responsible MU twitches. The results suggest that the afferent pathways mediating the mechanical events in muscle to MNs behave rather linearly, at least in the small-signal range of interest here.

Amplitude reduction of motor unit twitches during repetitive activation is accompanied by relative increase of hyperpolarizing membrane potential trajectories in homonymous alpha-motoneurons.

In anaesthetized cats, medial gastrocnemius motor units (MUs) were electrically stimulated via their ventral-root axons with independent random patterns. Isometric muscle tension and homonymous alpha-motoneuron (MN) membrane-potential fluctuations in response to these stimuli were recorded simultaneously, usually for periods of about 2 min. The tension and membrane potential were averaged with respect to a stimulus train over two disjoint time intervals, one stretching 20-40 s at record beginning, and the other a similar duration at the end of recording. Whereas average MU twitch amplitudes usually decreased between these periods, average membrane potential trajectories did not do so, such that, when normalized to the change in twitch amplitude, the membrane potential trajectories usually increased in size. This suggests that the decline in the mechanical effect of MU activation was accompanied by an increase in the gain of the afferent pathway to homonymous MNs, which was confirmed by gain computations in the frequency domain. This compensation could be a mechanism to maintain the high quality of information about MU contractions transmitted to MNs in the course of MU fatigue.

Effects of chloralose-urethane anesthesia on single-axon reciprocal Ia IPSPs in the cat.

Reciprocal Ia inhibitory postsynaptic potentials (IPSPs) generated by single afferents have been recorded with signal averaging in unanesthetized ischemic-decapitate cats for comparison with measurements previously obtained from preparations anesthetized with a mixture of chloralose and urethane. The results are similar to those which we obtained recently for single-axon recurrent IPSPs. Together, the studies show that chloralose-urethane anesthesia has a depressant effect on two widely studied circuits in the mammalian spinal cord.

Intramuscular localization of monosynaptic Ia reflex effects in the cat biceps femoris muscle.

Intracellular recordings form biceps femoris (BF) motoneurons were made in anesthetized low spinal cats during periods of electrical stimulation of the nerve branches supplying the anterior, middle and posterior portions of the BF muscle and the nerves to semimembranosus and semitendinosus. Measurements were made of each cell's composite intrahomonymous and heteronymous monosynaptic Ia-EPSP responses to stimulation of the test nerves (branches). We have found evidence for an intramuscular localization of these monosynaptic Ia reflex effects not only when comparing responses between the two functional components of the BF muscle as is well established [6] but, in addition, when comparing responses between different parts of each functional (hip extensor and knee flexor) component as well. It is argued that both somatotopic and neuronal recognition factors may contribute to the localization of these monosynaptic reflex effects.

Motor unit--muscle spindle interactions in active muscles of decerebrate cats.

Single muscle spindle afferent and motor unit EMG spike trains have been recorded simultaneously during periods of spontaneous motor activity in triceps surae muscles of decerebrate cats. The approximate time course and magnitude of the motor unit contractions were extracted from the whole muscle force record by spike-triggered averaging, and the functional interactions between motor unit contractions and spindle discharge were assessed by cross-correlating their respective spike trains. We have found that both spindle group Ia and II afferents are responsive to the contractions of single motor units in the presence of spontaneous motor activity, being strongly coupled to the activity of some motor units and indifferent to the contractions of others. Moreover, the cross-correlation analysis revealed modulation of a single motor unit's discharge pattern by the input of a single Ia afferent.

Computer-aided extraction of the features of the EMG of single motor units.

A software-based system is presented for feature extraction of compound, action-potential (EMG) recordings from single motor units. It simplifies and automates the measurement and analysis of several parameters of the action potential: peak-to-peak amplitude, total duration, peak-to-peak duration, and total area. The software is based on a simple algorithm that first finds the baseline (isoelectric line; including a noise level) of each single EMG potential (waveform) and then searches for the minimum and maximum values in the array of data points representing it. The algorithm searches in both directions starting from the minimum and maximum data points (the waveform peaks) to find the beginning and ending points of the waveform. Using the indices (i.e., array-point numbers) of the four data points provided by the algorithm, the desired features are extracted and/or calculated and saved in a standard-format spreadsheet. The algorithm has a potentially widespread usefulness in a broad array of electrophysiological studies.

Prolonged depression of force developed by single motor units after their intermittent activation in adult cats.

The fatigue of fast-twitch, glycolytic mammalian motor units [i.e., type FF; nomenclature of (3)] is dependent, in part, on the stimulation regimen (total number of stimuli, frequency, duty cycle, temporal patterning of stimuli, etc.) used to induce fatigue. To study the effect of the temporal pattern of the stimulus train on the rate and extend of fatigue in single FF units, one theoretically acceptable approach would be to use each motor unit as its own control: i.e., a sequential testing with two fatigue tests that differ only in the temporal organization of their stimuli. The purpose of this communication is to provide evidence that such an approach is not feasible when studying FF units, due to the delayed recovery of force following their repetitive activation. It was shown that 1/s activation of single FF units for only 15 or 45 s with intermittent 40-Hz, 300-ms duration trains significantly reduced their force response to a double-pulse shock for several hours. This finding suggests that in studies designed to test for the effects of different stimulation patterns on the fatigue of single motor units, deeply anaesthetized, reduced animal preparations are not appropriate models for the sequential application of different stimulation regimens to fast-twitch, glycolytic, mammalian motor units.

Distribution of sensory receptors in the flexor carpi radialis muscle of the cat.

The structures and distribution of encapsulated muscle receptors were examined in serial transverse sections of flexor carpi radialis in the adult cat. Four types of receptors (muscle spindles, Golgi tendon organs, paciniform, and Pacinian corpuscles) were identified. Their structures resembled those encountered in other limb muscles. Pacinian corpuscles were rare and occurred only in the external fascial coat of the muscle near its origin. The other three receptor types were distributed in an uneven but consistent pattern throughout the muscle. As noted previously (Gonyea and Ericson, '77), spindles were largely confined to a deep muscle region comprising less than 20% of the muscle volume, located directly between the long tendon of origin and the tendon of insertion. This region contains the largest proportion of type SO muscle fibers (Gonyea and Ericson, '77). Tendon organs and paciniform corpuscles were concentrated along the tendons that lined the spindle-rich muscle region. This region appeared to be composed of extrafusal fibers that were shorter and of more oblique pinnation than those in other regions. The localization of muscle receptors to the "oxidative" core of the muscle in its direct line of pull may have functional implications for afferent input to the spinal cord which are discussed. In addition, the possibility is raised that there are more paciniform corpuscles in flexor carpi radialis (and possibly other muscles) than previously thought.