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.

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.

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.

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.

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.

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.

Triggering module for waveform digitization.

A full circuit description is provided for a triggering module used to assist a small laboratory computer in digitizing muscle force- and EMG waveforms. During the stimulation of individual motor units using a standard fatigue test, a train of 13 pulses are delivered at a rate of 40 pps either intracellularly to a motor neuron, or extracellularly to functionally isolated single motor axons from among divided ventral-root nerve filaments. Trains are delivered at a rate of 1/s for the duration of the test, which may range from 120 to 3600 s. Both the force and EMG profiles undergo changes during such tests and the quantification of parameters associated with their waveforms are of interest to neurobiologists. The triggering module allows a typical small laboratory computer to capture user-selected waveforms and thereby reduces the programming problems, timing constraints, storage requirements and analysis time associated with obtaining these parameters. The versatile circuit may be easily adapted to solve similar data-acquisition problems. The method was implemented on an Apple Macintosh II computer but can also be applied to other systems equipped with appropriate software and a data-acquisition card.

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.

The motor units of cat medial gastrocnemius: electrical and mechanical properties as a function of muscle length.

The effects of changing muscle length on the mechanical properties of 89 motor units from adult cat medial gastrocnemius have been studied in eight experiments. Few differences were found between the effects of length on tetanic tension, twitch tension, twitch-tetanus ratio, twitch contraction time, twitch half relaxation time, rate of force development and electrical activity for fast contracting (twitch contraction time less than or equal to 45 msec) and slowly contracting (greater than 45 msec) units. Those differences that did appear did not persist when these two groups were matched by tetanic tension. It is concluded that the biophysical mechanisms responsible for the changes in mechanical and electrical properties with length must be similar for fast and slow twitch units and not related to potential differences in their muscle fiber type. The effects of changing muscle length on the mechanical properties of the eight whole muscles suggest that changes in force output with length are of minor importance during normal movements as the muscle is found to be electrically active over a relatively narrow range of lengths close to the optimum length for tetanus of the whole muscle. The very shortest muscle lengths at which there is only minimal force development are not used in natural movements, while the declining limb of the length tension curve is at muscle lengths beyond the maximum in situ length.

A commentary on muscle unit properties in cat hindlimb muscles.

A broad survey of muscle unit properties in 14 muscles of the cat hind limb is presented which emphasizes some general features of unit properties in mammalian muscles. A more detailed analysis of muscle unit properties in three muscles of the posterior compartment of the lower leg is then presented using Burke's tetrapartite (FF, FI or F (Int.), FR, and S) unit classification scheme. Our data on the properties of motor units in cat tibialis posterior (TP) have been compared to those generated by Burke and colleagues on units in flexor digitorum longus (FDL) and medial gastrocnemius (MG). In all three muscles, twitch contraction time was distinctly slower for type S units and specific tension outputs were substantially greater for type FF units than for type S units. The innervation ratios of type FR units were slightly lower than for type S units but the specific tension of the FR units was closer to FF units than to type S units. The FF units controlled 70-74% of the cumulative force output of each muscles, indicating a substantial capacity for powerful rapid contractions of all three of these muscles despite their differences in "size," action, and force generation. Distinctive features of the three muscles included differences in the unit types' force producing capabilities and in the relative representation of "nonfatigable" type FR and S units in each muscle. In particular, TP is endowed with some unusually powerful type FF units and a high percentage (42%) of type S units. In contrast, FDL has units that develop relatively little force and an unusually high representation (56%) of type FR units. The possible relationships between these muscle features and their presumed role in posture and locomotion is discussed.

Measurement systems calibration: microcomputer implementation.

Measurement systems used in the collection and processing of laboratory data must be calibrated periodically to obtain accurate results. Because calibration factors can change over time or may be reset to optimize measurements for specific tests, care must be taken to assure that calibration factors and data are aligned correctly. Users should be able to process current data or re-process older data using appropriate calibration factors. The alignment of calibration factors and data should occur in a simple, automatic and transparent way. This document describes one approach to calibration procedures and computer programs used to collect, process, document, measure and display laboratory data. The examples are from our neurophysiology laboratory, where investigators study the mammalian spinal cord and peripheral neuromuscular system. Typical calibration problems, some workable solutions, and computer programs (described in pseudocode) are presented.

Electromyographic responses of mammalian motor units to a fatigue test.

Evidence is presented that marked changes in the electromyographic (EMG) activity of single motor units often occur during a fatigue-test paradigm (12) widely used for the classification of mammalian motor units into fast-fatigable (FF), fast-intermediate-fatigable, fast-fatigue-resistant and slow categories (11), particularly in type FF units. Force output and EMG activity were measured in single motor units of the tibialis posterior muscle of anasthetized cats, while each unit was subjected to a fatigue test consisting of 4 min of motor-axon stimulation, using 1 Hz 330 ms trains of 0.1 ms shocks at 40 Hz. As a measure of the temporal characteristics of the EMG waveform, the reciprocal of the interval between first positive and subsequent negative peak was measured. For parameters of EMG magnitude, peak-to-peak amplitude and area were measured. The fatigue test was shown to produce, on average, significantly greater alterations in the values of the EMG parameters of FF units than of the other unit types. There were no significant EMG alterations among the other unit types. The results are discussed in relation to the interpretation of EMG depression as an indication of excitation failure and the relative fatigability and EMG depression of different motor unit types.

Cognitive set influences on Witkin's Rod-and-Frame Test.

Witkin's field-dependence theory is embroiled in conflict as research shows its primary assessment method, the Rod-and-frame Test, is influenced by situational events. This study explored the impact of an additional situational variable, locus of problem-solving data search, a cognitive set modified from Rotter's (1966) work. Two experimental groups of hospitalized alcoholics received standard Witkin instructions plus instructions emphasizing either an internal or external search for problem-solving cues. A control group received only Witkin's instructions. Aanlysis of variance showed the special instructions affected rod-and-frame scores in the predicted directions. The results were interpreted as indicating a need for a multi-factor approach to analyzing the complex relationships of the Rod-and-frame Test.