The in vitro neonatal rat spinal cord preparation: a new insight into mammalian locomotor mechanisms

The in vitro neonatal rat spinal cord preparation is the first mammalian nervous system isolated from the brainstem to the caudal end of the spinal cord. It permits the study of the cellular properties of mammalian locomotor networks and is unique in containing all the nervous structures related to locomotion. Although being a very immature system, this model has been considered as an adult preparation in which mammalian locomotor central pattern generators can be studied in detail. Nevertheless, one can also follow the development of locomotor functions during the perinatal period. Contrary to the adult, all neuroactive substances can directly reach the cellular structures in the brainstem-spinal cord preparation. When a neuroactive substance is applied to the bath, a single rhythmic activity is recorded along the cord. In fact, three rhythms can be isolated: one at the cervical level for the forelimbs, one at the lumbar level for the hind limbs and one in the sacrococcygeal region for the tail. Studies carried out on this preparation deal with three major areas: (1) relations between spontaneous activity and maturation of spinal network, (2) organisation of the different spinal networks, (3) key role of the descending pathways.Abbreviations 5-HT serotonin - ADP after-depolarization - AHP after-hyperpolarization - CPG central pattern generator - E0-E21 embryonic day 0–21 - INs interneurones - MLR mesencephalic locomotor region - MNs motoneurones - NMA N-methyl-d,l-aspartic acid - P0-P21 postnatal day 0–21 - PCPA p-chloro phenylalanin     

Spinal Mechanisms in the Control of Lamprey Swimming

SYNOPSIS. The lamprey, an anguilliform fish, swims using lateralundulatory movement; a transverse wave passes backward, fromhead to tail, the amplitude of the wave increasing as it movestailward. The wave of muscle activity producing this movementtravels down the body faster than the mechanical wave. The wayin which certain features of anguilliform movement contributeto its efficiency have been described. The neural activity underlyingswimming is characterized by: 1) rhythmical alternation betweenthe two sides of a single segment; 2) a burst duration thatremains a constant proportion of the cycle time and is independentof the cycle frequency; 3) rostrocaudal phase lag that is constantand also independent of the cycle frequency. Local circuitsin the lamprey spinal cord can generate this locomotory patternin the absence of sensory feedback following activation of excitatoryamino acid receptors; the pattern is centrally generated. Ithas been hypothesized that the spinal central pattern generatorfor locomotion consists of a series of segmental burst generatorscoupled together by an intersegmental coordinating system. Theintersegmental coordinating system functions to keep the frequenciesof the oscillators along the cord constant and to provide theappropriate rostrocaudal phase lag. Mechanosensitive units withinthe spinal cord are sensitive to movement of the spinal cord\notochordand movement of the spinal cord/notochord can entrain the burstpattern. Entrainment occurs through movement-related feedbackonto neurons at the local level. The possible roles this movement-relatedfeedback plays during locomotion are discussed.     

Intersegmental Coordination: Lessons from Modeling Systems of Coupled Non-Linear Oscillators

SYNOPSIS. Intersegmental coordination of both vertebrates andinvertebrates is poorly understood primarily because so littleis known about the substrate of the neural underpinnings andhow the elements interact to produce the complex timing relationshipsrequired by the organism. We describe here a systems approachcombining experimental and theoretical treatment of the lampreylocomotor central pattern generator. The central pattern generatoris viewed as a chain of coupled non-linear oscillators correspondingto segmental burst generators. We analyze various coupling schemesin terms of their ability to produce a stable traveling wavesimilar to that seen in the isolated spinal cord and the intactanimal. The role of long coordinating neurons is particularlydiscussed.     

Probing spinal circuits controlling walking in mammals

Locomotion in mammals is a complex motor act that involves the activation of a large number of muscles in a well-coordinated pattern. Understanding the network organization of the intrinsic spinal networks that control the locomotion, the central pattern generators, has been a challenge to neuroscientists. However, experiments using the isolated rodent spinal cord and combining electrophysiology and molecular genetics to dissect the locomotor network have started to shed new light on the network structure. In the present review, we will discuss findings that have revealed the role of designated populations of neurons for the key network functions including coordinating muscle activity and generating rhythmic activity. These findings are summarized in proposed organizational principles for the mammalian segmental CPG.     

Systems-level modeling of neuronal circuits for leech swimming

This paper describes a mathematical model of the neuronal central pattern generator (CPG) that controls the rhythmic body motion of the swimming leech. The systems approach is employed to capture the neuronal dynamics essential for generating coordinated oscillations of cell membrane potentials by a simple CPG architecture with a minimal number of parameters. Based on input/output data from physiological experiments, dynamical components (neurons and synaptic interactions) are first modeled individually and then integrated into a chain of nonlinear oscillators to form a CPG. We show through numerical simulations that the values of a few parameters can be estimated within physiologically reasonable ranges to achieve good fit of the data with respect to the phase, amplitude, and period. This parameter estimation leads to predictions regarding the synaptic coupling strength and intrinsic period gradient along the nerve cord, the latter of which agrees qualitatively with experimental observations.     

Neural network simulations of coupled locomotor oscillators in the lamprey spinal cord

The segmental locomotor network in the lamprey spinal cord was simulated on a computer using a connectionist-type neural network. The cells of the network were identical except for their excitatory levels and their synaptic connections. The synaptic connections used were based on previous experimental work. It was demonstrated that the connectivity of the circuit is capable of generating oscillatory activity with the appropriate phase relations among the cells. Intersegmental coordination was explored by coupling two identical segmental networks using only the cells of the network. Each of the possible couplings of a bilateral pair of cells in one oscillator with a bilateral pair of cells in the other oscillator produced stable phase locking of the two oscillators. The degree of phase difference was dependent upon synaptic weight, and the operating range of synaptic weights varied among the pairs of connections. The coupling was tested using several criteria from experimental work on the lamprey spinal cord. Coupling schemes involving several pairs of connecting cells were found which 1) achieved steadystate phase locking within a single cycle, 2) exhibited constant phase differences over a wide range of cycle periods, and 3) maintained stable phase locking in spite of large differences in the intrinsic frequencies of the two oscillators. It is concluded that the synaptic connectivity plays a large role in producing oscillations in this network and that it is not necessary to postulate a separate set of coordinating neurons between oscillators in order to achieve appropriate phase coupling.     

Estimating the Strength and Direction of Functional Coupling in the Lamprey Spinal Cord

A method of estimating coupling strength between two neural oscillators based on their spikes trains (Kiemel and Cohen, J. Comput. Neurosci. 5: 267–284, 1998) is tested using simulated data and then applied to experimental data from the central pattern generator (CPG) for swimming in the lamprey. The method is tested using a model of two connectionist oscillators and a model of two endogenously bursting cells. For both models, the method provides useful estimates of the relative strength of coupling in each direction, as well as estimates of total strength. The method is applied to pairs of motor-nerve recordings from isolated 50-segment pieces of spinal cords from adult silver lampreys (Ichthyomyzon unicuspus). The strength and direction of coupling is estimated under control conditions and conditions in which intersegmental coupling between the two recording locations is weakened by hemisections of the spinal cords and/or chambers containing an inhibitory solution that blocks firing in postsynaptic cells. The relevance of these measures in constraining models of the CPG is discussed.     

Modeling of a bipedal robot using mutually coupled Rayleigh oscillators

The objective of the work presented here was the modeling of a bipedal robot using a central pattern generator (CPG) formed by a set of mutually coupled Rayleigh oscillators. We analyzed a 2D model, with the three most important determinants of gait, that performs only motions parallel to the sagittal plane. Using oscillators with integer relation of frequency, we determined the transient motion and the stable limit cycles of the network formed by the three oscillators, showing the behavior of the knee angles and the hip angle. A comparison of the plotted graphs revealed that the system provided excellent results when compared to experimental analysis. Based on the results of the study, we come to the conclusion that the use of mutually coupled Rayleigh oscillators can represent an excellent method of signal generation, allowing their application for feedback control of a walking machine.Acknowledgements The authors would like to express their gratitude to CNPq and CAPES for the financial support provided during the course of this research.     

Expression of protocadherin-9 and protocadherin-17 in the nervous system of the embryonic zebrafish

In this study we analyzed expression patterns of two δ-protocadherins, protocadherin-9 and protocadherin-17, in the developing zebrafish using in situ hybridization and RT-PCR methods. Both protocadherins were mainly detected in the embryonic central nervous system, but each showed a distinct expression pattern. Protocadherin-9 message (Pcdh9) was expressed after 10 h post fertilization (hpf). It was found mainly in small clusters of cells in the anteroventral forebrain and ventrolateral hindbrain, and scattered cells throughout the spinal cord of young embryos (24 hpf). Pcdh9 expression in the hindbrain was segmental, reflecting a neuromeric organization, which became more evident at 34 hpf. As development proceeded, Pcdh9 expression increased throughout the brain, while its expression in the spinal cord was greatly reduced. Pcdh9 was also found in the developing retina and statoacoustic ganglion. Protocadherin-17 message (Pcdh17) expression began much earlier (1.5–2 hpf) than Pcdh9. Similar to Pcdh9 expression, Pcdh17 expression was found mainly in the anteroventral forebrain at 24 hpf, but its expression in the hindbrain and spinal cord, confined mainly to lateroventral regions of the hindbrain and anterior spinal cord, was more restricted than Pcdh9. As development proceeded, Pcdh17 expression was increased both in the brain and spinal cord: detected throughout the brain of two- and three-day old embryos, strongly expressed in the retina and in lateral regions of spinal cord in two-day old embryos. Its expression in the retina and spinal cord was reduced in three-day old embryos. Our results showed that expression of these two protocadherins was both spatially and temporally regulated.     

The neuroethology of sound production in tiger moths (Lepidoptera,Arctiidae)

When stimulated either acoustically or tactually, certain species of arctiid moths rhythmically emit trains of clicks from metathoracic tymbals. The purpose of the experiments presented here was to determine the location within the central nervous system (CNS) of the proposed tymbal central pattern generator (CPG) in Cycnia tenera. Motor neuron impulses that underlie tymbal activation were recorded extracellularly from the tymbal nerve while moths were subjected to selective severing of the suboesophageal, prothoracic, pterothoracic and abdominal ganglia connectives. Motor output evoked by either acoustic or tactile stimulation originates from a common CPG because tymbal nerve spikes in both cases are similar in amplitude, waveform and rhythmicity. Our results showed: (1) removal of the CNS posterior of the second abdominal neuromere had no effect, (2) removal of the head decreased the responsiveness of the animal to acoustic stimulation and, (3) severing the connectives between the prothoracic and pterothoracic ganglia abolished responses to acoustic stimuli and diminished responses to tactile stimuli. We conclude that although the minimal circuitry sufficient for activating the tymbals resides in the pterothoracic ganglion, the prothoracic and cephalic ganglia are required for the normal, and in particular, auditory-evoked operation of the tymbal CPG.Abbreviations ASR acoustic startle response - CNS central nervous system - CPG central pattern generator - dB peSPL decibel peak equivalent sound pressure level (rms re 20 Pa) - ISI inter-spike interval     

Activity-dependent modulation of adaptation produces a constant burst proportion in a model of the lamprey spinal locomotor generator

The neuronal network underlying lamprey swimming has stimulated extensive modelling on different levels of abstraction. The lamprey swims with a burst frequency ranging from 0.3 to 8–10 Hz with a rostro-caudal lag between bursts in each segment along the spinal cord. The swimming motor pattern is characterized by a burst proportion that is independent of burst frequency and lasts around 30%–40% of the cycle duration. This also applies in preparations in which the reciprocal inhibition in the spinal cord between the left and right side is blocked. A network of coupled excitatory neurons producing hemisegmental oscillations may form the basis of the lamprey central pattern generator (CPG). Here we explored how such networks, in principle, could produce a large frequency range with a constant burst proportion. The computer simulations of the lamprey CPG use simplified, graded output units that could represent populations of neurons and that exhibit adaptation. We investigated the effect of an active modulation of the degree of adaptation of the CPG units to accomplish a constant burst proportion over the whole frequency range when, in addition, each hemisegment is assumed to be self-oscillatory. The degree of adaptation is increased with the degree of stimulation of the network. This will make the bursts terminate earlier at higher burst rates, allowing for a constant burst proportion. Without modulated adaptation the network operates in a limited range of swimming frequencies due to a progressive increase of burst duration with increasing background stimulation. By introducing a modulation of the adaptation, a broad burst frequency range can be produced. The reciprocal inhibition is thus not the primary burst terminating factor, as in many CPG models, and it is mainly responsible for producing alternation between the left and right sides. The results are compared with the Morris-Lecar oscillator model with parameters set to produce a type A and type B oscillator, in which the burst durations stay constant or increase, respectively, when the background stimulation is increased. Here as well, burst duration can be controlled by modulation of the slow variable in a similar way as above. When oscillatory hemisegmental networks are coupled together in a chain a phase lag is produced. The production of a phase lag in chains of such oscillators is compared with chains of Morris-Lecar relaxation oscillators. Models relating to the intact versus isolated spinal cord preparation are discussed, as well as the role of descending inhibition. Received: 1 April 1997 / Accepted in revised form: 20 March 1998     

Synaptic patterning of left-right alternation in a computational model of the rodent hindlimb central pattern generator

Establishing, maintaining, and modifying the phase relationships between extensor and flexor muscle groups is essential for central pattern generators in the spinal cord to coordinate the hindlimbs well enough to produce the basic walking rhythm. This paper investigates a simplified computational model for the spinal hindlimb central pattern generator (CPG) that is abstracted from experimental data from the rodent spinal cord. This model produces locomotor-like activity with appropriate phase relationships in which right and left muscle groups alternate while extensor and flexor muscle groups alternate. Convergence to this locomotor pattern is slow, however, and the range of parameter values for which the model produces appropriate output is relatively narrow. We examine these aspects of the model’s coordination of left-right activity through investigation of successively more complicated subnetworks, focusing on the role of the synaptic architecture in shaping motoneuron phasing. We find unexpected sensitivity in the phase response properties of individual neurons in response to stimulation and a need for high levels of both inhibition and excitation to achieve the walking rhythm. In the absence of cross-cord excitation, equal levels of ipsilateral and contralateral inhibition result in a strong preference for hopping over walking. Inhibition alone can produce the walking rhythm, but contralateral inhibition must be much stronger than ipsilateral inhibition. Cross-cord excitatory connections significantly enhance convergence to the walking rhythm, which is achieved most rapidly with strong crossed excitation and greater contralateral than ipsilateral inhibition. We discuss the implications of these results for CPG architectures based on unit burst generators.     

The role of afferent feedback in the control of hamstrings activity during human gait

In vertebrates, possibly also in man, the pattern of activation of muscles during locomotion can be generated by the spinal cord (locomotor CPG, central pattern generator). However, sensory feedback is crucial to adapt the functioning of the CPG to the external requirements during gait. It is postulated that afferent input from skin and muscles can contribute to the EMG activation patterns as observed in various limb muscles during gait. The activity of the hamstrings at end swing may be partially due to stretch reflexes of these muscles. At end stance the hamstring activity may be assisted by reflexes from natural skin activation from the dorsum of the foot. In addition, more specific actions are also incorporated. For example, sural nerve stimulation induces an activation of biceps femoris (BF) whereas a suppression is usually obtained for semitendinosus (ST), indicating that the induced activation is aimed at exorotation of the lower leg. Similarly, the preferential activation of medial versus lateral gastrocnemius (GM versus GL) in sural nerve induced reflexes could favor such exorotation. It is concluded that the present evidence points towards a possible contribution of various reflexes to the motor output seen during gait for movements both inside and outside the sagittal plane.     

The Calculation of Frequency-Shift Functions for Chains of Coupled Oscillators, with Application to a Network Model of the Lamprey Locomotor Pattern Generator

Chains of coupled limit-cycle oscillators are considered, in which the coupling is assumed to be weak and only between adjacent oscillators. For such a system the change in frequency of an oscillator due to the coupling can be expressed, up to first order in thecoupling strength, by functions that depend only on the phase difference between the coupled oscillators. In this article a numerical algorithm is developed for the evaluation of these functions (the H-functions) in terms of a single oscillator and the interactions between coupled oscillators. The technique is applied to a connectionist model for the locomotor pattern generator in the lamprey spinal cord.An H-function so derived is compared to a function derived empirically(the C-function) from simulations of the same system. The phase lagsthat develop between adjacent oscillators in a simulated chain are compared with those predicted theoretically, and it is shown that coupling thatis functionally strong is nonetheless weak enough to behave as predicted.     

Neural Control of Interappendage Phase During Locomotion

Interappendage phasing of crayfish swimmeret movements dependsupon a central nervous system network of command, oscillator,and coordinating neurons. The command neurons serve to set thegeneral excitation level in each of the segmental oscillators.The oscillator neurons in each hemi-ganglion generate the rhythmicalternations of powerstroke and returnstroke motor neuron activity.The coordinating neurons transmit the precise timing informationabout the state of one oscillator to other oscillators. Thisinformation can serve to advance or to delay the motor burstsdriven by the other oscillators. Which effect is observed dependsupon the arrival time of the coordinating neuron discharge withinthe cycle period of the modulated oscillator. This type of modulationleads to the prediction that a stable interappendage phase canresult from situations where there is not a fixed excitabilitygradient among the segmental oscillators. This prediction hasbeen verified using a cut command neuron preparation.     

Hard-wired central pattern generators for quadrupedal locomotion

Animal locomotion is generated and controlled, in part, by a central pattern generator (CPG), which is an intraspinal network of neurons capable of producing rhythmic output. In the present work, it is demonstrated that a hard-wired CPG model, made up of four coupled nonlinear oscillators, can produce multiple phase-locked oscillation patterns that correspond to three common quadrupedal gaits — the walk, trot, and bound. Transitions between the different gaits are generated by varying the network's driving signal and/or by altering internal oscillator parameters. The above in numero results are obtained without changing the relative strengths or the polarities of the system's synaptic interconnections, i.e., the network maintains an invariant coupling architecture. It is also shown that the ability of the hard-wired CPG network to produce and switch between multiple gait patterns is a model-independent phenomenon, i.e., it does not depend upon the detailed dynamics of the component oscillators and/or the nature of the inter-oscillator coupling. Three different neuronal oscillator models — the Stein neuronal model, the Van der Pol oscillator, and the FitzHugh-Nagumo model -and two different coupling schemes are incorporated into the network without impeding its ability to produce the three quadrupedal gaits and the aforementioned gait transitions.     

GABA regulates the buccal motor output of Helisoma by two pharmacologically distinct actions

GABA was tested for its effects on patterned motor activity (PMA) underlying feeding. Using buccal motoneuron B19 to monitor PMA through intracellular recordings, GABA was found to exert effects at two levels. First, GABA stimulated rhythmic patterned activity resembling fictive feeding, which is under the control of the buccal CPG. In addition, GABA produced a direct inhibition of neuron B19. Both effects were observed when the buccal ganglia were studied in isolation from the rest of the central nervous system, suggesting local interactions with GABA receptors of buccal neurons. Furthermore, these two actions of GABA were found to be pharmacologically distinguishable. The direct hyperpolarization of neuron B19 was mimicked by muscimol, but not baclofen, and involved an increased chloride conductance, which was blocked by picrotoxin.Baclofen duplicated CPG activation by GABA. Picrotoxin had no effect on GABA- or baclofen-induced PMA.These results demonstrate that the Helisoma buccal ganglia have two GABA receptor types which resemble, pharmacologically, mammalian GABA_A and GABA_B receptors, and that GABA plays a key role in feeding patterned motor activity in Helisoma.Abbreviations CPG central pattern generator - GABA gammaamino butyric acid - HPLC high performance liquid chromatography - IPSP inhibitory postsynaptic potential - PMA patterned motor activity - SLRT supralateral radular tensor muscle     

Multi-layered multi-pattern CPG for adaptive locomotion of humanoid robots

In this paper, we present an extended mathematical model of the central pattern generator (CPG) in the spinal cord. The proposed CPG model is used as the underlying low-level controller of a humanoid robot to generate various walking patterns. Such biological mechanisms have been demonstrated to be robust in locomotion of animal. Our model is supported by two neurophysiological studies. The first study identified a neural circuitry consisting of a two-layered CPG, in which pattern formation and rhythm generation are produced at different levels. The second study focused on a specific neural model that can generate different patterns, including oscillation. This neural model was employed in the pattern generation layer of our CPG, which enables it to produce different motion patterns—rhythmic as well as non-rhythmic motions. Due to the pattern-formation layer, the CPG is able to produce behaviors related to the dominating rhythm (extension/flexion) and rhythm deletion without rhythm resetting. The proposed multi-layered multi-pattern CPG model (MLMP-CPG) has been deployed in a 3D humanoid robot (NAO) while it performs locomotion tasks. The effectiveness of our model is demonstrated in simulations and through experimental results.     

Longitudinal coordination of motor output during swimming in Xenopus embryos.

Little is known about the neural mechanisms that control the phenomenon of rostro-caudal delay. In Xenopus embryos there is a constant rostro-caudal delay of 2-5 ms mm-1 during fictive swimming. Rostro-caudal delay is not significantly correlated with cycle period. When NMDA is applied to the caudal spinal cord there is a decrease and in some cases a reversal in rostro-caudal delay. Conversely applying excitatory antagonists to the caudal spinal cord leads to an increase in delay. When caudal mid-cycle inhibition is reduced either pharmacologically using strychnine or surgically through hemisection of the spinal cord, there is an increase in rostro-caudal delay. Rostro-caudal delays are too small to be explainable on the basis of axonal conduction velocities and synaptic delays. This suggests that the central pattern generator of Xenopus behaves as a series of coupled oscillators and that the nature of the coupling, together with a longitudinal gradient in excitability associated with the oscillators, contributes to the observed rostro-caudal delay.