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1.  Modeling the mammalian locomotor CPG: insights from mistakes and perturbations 
Progress in brain research  2007;165:235-253.
A computational model of the mammalian spinal cord circuitry incorporating a two-level central pattern generator (CPG) with separate half-center rhythm generator (RG) and pattern formation (PF) networks is reviewed. The model consists of interacting populations of interneurons and motoneurons described in the Hodgkin-Huxley style. Locomotor rhythm generation is based on a combination of intrinsic (persistent sodium current dependent) properties of excitatory RG neurons and reciprocal inhibition between the two half-centers comprising the RG. The two-level architecture of the CPG was suggested from an analysis of deletions (spontaneous omissions of activity) and the effects of afferent stimulation on the locomotor pattern and rhythm observed during fictive locomotion in the cat. The RG controls the activity of the PF network that in turn defines the rhythmic pattern of motoneuron activity. The model produces realistic firing patterns of two antagonist motoneuron populations and generates locomotor oscillations encompassing the range of cycle periods and phase durations observed during cat locomotion. A number of features of the real CPG operation can be reproduced with separate RG and PF networks, which would be difficult if not impossible to demonstrate with a classical single-level CPG. The two-level architecture allows the CPG to maintain the phase of locomotor oscillations and cycle timing during deletions and during sensory stimulation. The model provides a basis for functional identification of spinal interneurons involved in generation and control of the locomotor pattern.
PMCID: PMC2408748  PMID: 17925250
spinal cord; CPG; rhythm generation; locomotion; afferent control
2.  Some principles of organization of spinal neurons underlying locomotion in zebrafish and their implications 
Recent studies of the spinal motor system of zebrafish, along with work in other species, are leading to some principles that appear to underlie the organization and recruitment of motor networks in cord: (1) broad neuronal classes defined by a set of transcription factors, key morphological features, and transmitter phenotypes arise in an orderly way from different dorso-ventral zones in spinal cord; (2) motor behaviors and both motoneurons and interneurons differentiate in order from gross, often faster, movements and the neurons driving them to progressively slower movements and their underlying neurons; (3) recruitment order of motoneurons and interneurons is based upon time of differentiation; (4) different locomotor speeds involve some shifts in the set of active interneurons. Here we review these principles and some of their implications for other parts of the brain, other vertebrates, and limbed locomotion.
PMCID: PMC3579554  PMID: 20536924
motoneurons; spinal interneurons; transcription factors; locomotion; motor pattern
3.  Cholinergic Partition Cells and Lamina X Neurons Induce a Muscarinic-Dependent Short-Term Potentiation of Commissural Glutamatergic Inputs in Lumbar Motoneurons 
Acetylcholine and the activation of muscarinic receptors influence the activity of neural networks generating locomotor behavior in the mammalian spinal cord. Using electrical stimulations of the ventral commissure, we show that commissural muscarinic (CM) depolarizations could be induced in lumbar motoneurons. We provide a detailed electrophysiological characterization of the muscarinic receptors and the membrane conductance involved in these responses. Activation of the CM terminals, originating from lamina X neurons and partition cells, induced a pathway-specific short-term potentiation (STP) of commissural glutamatergic inputs in motoneurons. This STP is occluded in the presence of the muscarinic antagonist atropine. During fictive locomotion, the activation of the commissural pathways transiently enhanced the motor output in a muscarinic-dependent manner. This study describes for the first time a novel regulatory mechanism of synaptic strength in spinal locomotor networks. Such cellular mechanisms would endow the locomotor central pattern generators with adaptive processes needed to generate appropriate synaptic inputs to motoneurons during different motor tasks.
PMCID: PMC3208176  PMID: 22069380
motoneurons; muscarinic-dependent-short-term potentiation/modulation of synaptic transmission; commissural cholinergic interneurons
4.  Spinal cord injury induces serotonin supersensitivity without increasing intrinsic excitability of mouse V2a interneurons 
Denervation induced plastic changes impair locomotor recovery after spinal cord injury (SCI). Spinal motoneurons become hyperexcitable after SCI, but the plastic responses of locomotor network interneurons after SCI have not been studied. Using an adult mouse SCI model, we analyzed the effects of complete spinal cord lesions on the intrinsic electrophysiological properties, excitability and neuromodulatory responses to serotonin (5-HT) in mouse lumbar V2a spinal interneurons, which help regulate left-right alternation during locomotion. Four weeks after SCI, V2a interneurons showed almost no changes in baseline excitability or action potential properties; the only parameter that changed was a reduced input resistance. However, V2a interneurons became 100-1000 fold more sensitive to 5-HT. Immunocytochemical analysis showed that SCI caused a coordinated loss of serotonergic fibers and the 5-HT transporter (SERT). Blocking the SERT with citalopram in intact mice did not increase 5-HT sensitivity to the level seen after SCI. SCI also evoked an increase in 5-HT2C receptor cluster number and intensity, suggesting that several plastic changes cooperate in increasing 5-HT sensitivity. Our results suggest that different components of the spinal neuronal network responsible for coordinating locomotion are differentially affected by SCI, and highlight the importance of understanding these changes when considering therapies targeted at functional recovery.
PMCID: PMC3506248  PMID: 22993431
5.  Mechanisms of excitation of spinal networks by stimulation of the ventral roots 
It has recently been demonstrated that motoneurons in neonatal rodents release an excitatory amino acid, in addition to acetylcholine, from their central terminals onto Renshaw cells. Although the function of this amino acid release is not understood, it may mediate the excitatory actions of motor axon stimulation on spinal motor networks. Stimulation of motor axons in the ventral roots or muscle nerves can activate the locomotor central pattern generator or entrain bursting in the disinhibited cord. Both of these effects persist in the presence of cholinergic antagonists and are abolished or diminished by ionotropic and metabotropic glutamate antagonists.
Calcium imaging in the disinhibited cord shows that a ventral root stimulus evokes ventrolateral activity initially which subsequently propagates to the rest of the cord. This finding suggests that excitatory interneurons excited by motoneuron recurrent collaterals are located in this region. However, motoneurons do not exhibit short latency excitatory potentials in response to ventral root stimulation indicating that the excitatory effects are mediated polysynaptically. The significance of these findings is discussed.
PMCID: PMC3033581  PMID: 20536921
calcium imaging; motoneuron; recurrent excitation; spinal cord
6.  Somato-dendritic morphology and dendritic signal transfer properties differentiate between fore- and hindlimb innervating motoneurons in the frog Rana esculenta 
BMC Neuroscience  2012;13:68.
The location specific motor pattern generation properties of the spinal cord along its rostro-caudal axis have been demonstrated. However, it is still unclear that these differences are due to the different spinal interneuronal networks underlying locomotions or there are also segmental differences in motoneurons innervating different limbs. Frogs use their fore- and hindlimbs differently during jumping and swimming. Therefore we hypothesized that limb innervating motoneurons, located in the cervical and lumbar spinal cord, are different in their morphology and dendritic signal transfer properties. The test of this hypothesis what we report here.
Discriminant analysis classified segmental origin of the intracellularly labeled and three-dimensionally reconstructed motoneurons 100% correctly based on twelve morphological variables. Somata of lumbar motoneurons were rounder; the dendrites had bigger total length, more branches with higher branching orders and different spatial distributions of branch points. The ventro-medial extent of cervical dendrites was bigger than in lumbar motoneurons. Computational models of the motoneurons showed that dendritic signal transfer properties were also different in the two groups of motoneurons. Whether log attenuations were higher or lower in cervical than in lumbar motoneurons depended on the proximity of dendritic input to the soma. To investigate dendritic voltage and current transfer properties imposed by dendritic architecture rather than by neuronal size we used standardized distributions of transfer variables. We introduced a novel combination of cluster analysis and homogeneity indexes to quantify segmental segregation tendencies of motoneurons based on their dendritic transfer properties. A segregation tendency of cervical and lumbar motoneurons was detected by the rates of steady-state and transient voltage-amplitude transfers from dendrites to soma at all levels of synaptic background activities, modeled by varying the specific dendritic membrane resistance. On the other hand no segregation was observed by the steady-state current transfer except under high background activity.
We found size-dependent and size-independent differences in morphology and electrical structure of the limb moving motoneurons based on their spinal segmental location in frogs. Location specificity of locomotor networks is therefore partly due to segmental differences in motoneurons driving fore-, and hindlimbs.
PMCID: PMC3472316  PMID: 22708833
7.  Organization of mammalian locomotor rhythm and pattern generation 
Brain research reviews  2007;57(1):134-146.
Central pattern generators (CPGs) located in the spinal cord produce the coordinated activation of flexor and extensor motoneurons during locomotion. Previously proposed architectures for the spinal locomotor CPG have included the classical half-center oscillator and the unit burst generator (UBG) comprised of multiple coupled oscillators. We have recently proposed another organization in which a two-level CPG has a common rhythm generator (RG) that controls the operation of the pattern formation (PF) circuitry responsible for motoneuron activation. These architectures are discussed in relation to recent data obtained during fictive locomotion in the decerebrate cat. The data show that the CPG can maintain the period and phase of locomotor oscillations both during spontaneous deletions of motoneuron activity and during sensory stimulation affecting motoneuron activity throughout the limb. The proposed two-level CPG organization has been investigated with a computational model which incorporates interactions between the CPG, spinal circuits and afferent inputs. The model includes interacting populations of spinal interneurons and motoneurons modeled in the Hodgkin-Huxley style. Our simulations demonstrate that a relatively simple CPG with separate RG and PF networks can realistically reproduce many experimental phenomena including spontaneous deletions of motoneuron activity and a variety of effects of afferent stimulation. The model suggests plausible explanations for a number of features of real CPG operation that would be difficult to explain in the framework of the classical single-level CPG organization. Some modeling predictions and directions for further studies of locomotor CPG organization are discussed.
PMCID: PMC2214837  PMID: 17936363
CPG; computational models; spinal cord; decerebrate; cat
8.  A Gradient in Endogenous Rhythmicity and Oscillatory Drive Matches Recruitment Order in an Axial Motor Pool 
The rhythmic firing behavior of spinal motoneurons is a function of their electrical properties and synaptic inputs. However, the relative contribution of endogenous versus network-based rhythmogenic mechanisms to locomotion is unclear. To address this issue, we have recorded from identified motoneurons and compared their current-evoked firing patterns to network-driven ones in the larval zebrafish (Danio rerio). Zebrafish axial motoneurons are recruited topographically from the bottom of the spinal cord up. Here, we have explored differences in the morphology of axial motoneurons, their electrical properties and their synaptic drive, to reveal how they match the topographic pattern of recruitment. More ventrally located ‘secondary’ motoneurons generate bursts of action potentials in response to constant current steps, demonstrating a strong inherent rhythmogenesis. The membrane potential oscillations underlying bursting behavior occur in the normal frequency range of swimming. In contrast, more dorsal secondaries chatter in response to current, while the most dorsally distributed ‘primary’ motoneurons all fire tonically. We find that systematic variations in excitability and endogenous rhythmicity are inversely related to the level of oscillatory synaptic drive within the entire axial motor pool. Specifically, bursting cells exhibit the least amount of drive while tonic cells exhibit the most. Our data suggest that increases in swimming frequency are accomplished by the recruitment of axial motoneurons that progressively rely on instructive synaptic drive to shape their oscillatory activity appropriately. Thus, within the zebrafish spinal cord there are differences in the relative contribution of endogenous versus network-based rhythms to locomotion and these vary predictably according to order of recruitment.
PMCID: PMC3428065  PMID: 22875927
9.  A cluster of cholinergic pre-motor interneurons modulates mouse locomotor activity 
Neuron  2009;64(5):645-662.
Mammalian motor programs are controlled by networks of spinal interneurons that set the rhythm and intensity of motor neuron firing. Motor neurons have long been known to receive prominent ‘C-bouton’ cholinergic inputs from spinal interneurons, but the source and function of these synaptic inputs have remained obscure. We show here that the transcription factor Pitx2 marks a small cluster of spinal cholinergic interneurons, V0C neurons, that represents the sole source of C-bouton inputs to motor neurons. The activity of these cholinergic interneurons is tightly phase-locked with motor neuron bursting during fictive locomotor activity, suggesting a role in the modulation of motor neuron firing frequency. Genetic inactivation of the output of these neurons impairs a locomotor task-dependent increase in motor neuron firing and muscle activation. Thus V0C interneurons represent a defined class of spinal cholinergic interneurons with an intrinsic neuromodulatory role in the control of locomotor behavior.
PMCID: PMC2891428  PMID: 20005822
cholinergic interneurons; synapses; locomotor activity; neuromodulation
10.  Spinal interneuronal networks in the cat; elementary components 
Brain research reviews  2007;57(1):46-55.
This review summarises features of networks of commissural interneurones co-ordinating muscle activity on both sides of the body as an example of feline elementary spinal interneuronal networks. The main feature of these elementary networks is that they are interconnected and incorporated into more complex networks as their building blocks. Links between networks of commissural interneurones and other networks are quite direct, with mono- and disynaptic input from the reticulospinal and vestibulospinal neurones, disynaptic from the contralateral and ipsilateral corticospinal neurones and fastigial neurones, di- or oligosynaptic from the mesencephalic locomotor region and mono-, di- or oligosynaptic from muscle afferents. The most direct links between commissural interneurones and motoneurones are likewise simple: monosynaptic and disynaptic via premotor interneurones with input from muscle afferents. By such connections a particular elementary interneuronal network may subserve a wide range of movements, from simple reflex and postural adjustments to complex centrally initiated phasic and rhythmic movements, including voluntary movements and locomotion. Other common features of the commissural and other interneuronal networks investigated so far is that input from several sources is distributed to their constituent neurones in a semi-random fashion and that there are several possibilities of interactions between neurones both within and between various populations. Neurones of a particular elementary network are located at well defined sites but intermixed with neurones of other networks and distributed over considerable lengths of the spinal cord, which precludes the topography to be used as their distinguishing feature.
PMCID: PMC2683333  PMID: 17884173
Spinal cord; interneuron; commissural neuron; networks
11.  Persistent inward currents in spinal motoneurons: Important for normal function but potentially harmful after spinal cord injury and in amyotrophic lateral sclerosis 
Meaningful body movements depend on the interplay between synaptic inputs to motoneurons and their intrinsic properties. Injury and disease often alter either or both of these factors and cause motoneuron and movement dysfunction. The ability of the motoneuronal membrane to generate persistent inward currents (PICs) is especially potent in setting the intrinsic excitability of motoneurons and can drastically change the motoneuron output to a given input. In this article, we review the role of PICs in modulating the excitability of spinal motoneurons during health, and their contribution to motoneuron excitability after spinal cord injury (SCI) and in amyotrophic lateral sclerosis (ALS) leading to exaggerated long-lasting reflexes and muscle spasms, and contributing to neuronal degeneration, respectively.
PMCID: PMC3000632  PMID: 20462789
Motor neuron; Motoneuron; Persistent inward current; Amyotrophic lateral sclerosis; Spinal cord injury
12.  The Use of PRV-Bartha to Define Premotor Inputs to Lumbar Motoneurons in the Neonatal Spinal Cord of the Mouse 
PLoS ONE  2010;5(7):e11743.
The neonatal mouse has become a model system for studying the locomotor function of the lumbar spinal cord. However, information about the synaptic connectivity within the governing neural network remains scarce. A neurotropic pseudorabies virus (PRV) Bartha has been used to map neuronal connectivity in other parts of the nervous system, due to its ability to travel trans-neuronally. Its use in spinal circuits regulating locomotion has been limited and no study has defined the time course of labelling for neurons known to project monosynaptically to motoneurons.
Methodology/Principal Findings
Here we investigated the ability of PRV Bartha, expressing green and/or red fluorescence, to label spinal neurons projecting monosynaptically to motoneurons of two principal hindlimb muscles, the tibialis anterior (TA) and gastrocnemius (GC). As revealed by combined immunocytochemistry and confocal microscopy, 24–32 h after the viral muscle injection the label was restricted to the motoneuron pool while at 32–40 h the fluorescence was seen in interneurons throughout the medial and lateral ventral grey matter. Two classes of ipsilateral interneurons known to project monosynaptically to motoneurons (Renshaw cells and cells of origin of C-terminals) were consistently labeled at 40 h post-injection but also a group in the ventral grey matter contralaterally. Our results suggest that the labeling of last order interneurons occurred 8–12 h after motoneuron labeling and we presume this is the time taken by the virus to cross one synapse, to travel retrogradely and to replicate in the labeled cells.
The study establishes the time window for virally - labelling monosynaptic projections to lumbar motoneurons following viral injection into hindlimb muscles. Moreover, it provides a good foundation for intracellular targeting of the labeled neurons in future physiological studies and better understanding the functional organization of the lumbar neural networks.
PMCID: PMC2909228  PMID: 20668534
13.  Spinal Commissural Connections to Motoneurons Controlling the Primate Hand and Wrist 
The Journal of Neuroscience  2013;33(23):9614-9625.
Left–right coordination is essential for locomotor movements and is partly mediated by spinal commissural systems. Such coordination is also essential for reaching and manipulation in primates, but the role of spinal commissural systems here has not been studied. We investigated commissural connectivity to motoneurons innervating forelimb muscles using intracellular recordings in acutely anesthetized macaque monkeys. In 57 of 81 motoneurons, synaptic responses (52 of 57 excitatory) were evoked after contralateral intraspinal microstimulation in the gray matter (cISMS; 300 μA maximum current intensity). Some responses (15 of 57) occurred at latencies compatible with a monosynaptic linkage, including in motoneurons projecting to intrinsic hand muscles (9 cells). Three pieces of evidence suggest that these effects reflected the action of commissural interneurons. In two cells, preceding cISMS with stimulation of the contralateral medial brainstem descending pathways facilitated the motoneuron responses, suggesting that cISMS acted on cell bodies whose excitability was increased by descending inputs. Pairing cISMS with stimulation of the contralateral corticospinal tract yielded no evidence of response occlusion in 16 cells tested, suggesting that the effects were not merely axon reflexes generated by stimulation of corticospinal axon branches, which cross the midline. Finally, stimulation of contralateral peripheral nerves evoked responses in 28 of 52 motoneurons (7 of 9 projecting to the hand). Our results demonstrate the existence of commissural neurons with access to spinal motoneurons in primate cervical spinal cord that receive inputs from the periphery as well as descending pathways. Most importantly, commissural neurons also innervate motoneurons of intrinsic hand muscles.
PMCID: PMC3951829  PMID: 23739958
14.  Glutamatergic Mechanisms for Speed Control and Network Operation in the Rodent Locomotor CPG 
Locomotion is a fundamental motor act that, to a large degree, is controlled by central pattern-generating (CPG) networks in the spinal cord. Glutamate is thought to be responsible for most of the excitatory input to and the excitatory activity within the locomotor CPG. However, previous studies in mammals have produced conflicting results regarding the necessity and role of the different ionotropic glutamate receptors (GluRs) in the CPG function. Here, we use electrophysiological and pharmacological techniques in the in vitro neonatal mouse lumbar spinal cord to investigate the role of a broad range of ionotropic GluRs in the control of locomotor speed and intrinsic locomotor network function. We show that non-NMDA (non-NMDARs) and NMDA receptor (NMDAR) systems may independently mediate locomotor-like activity and that these receptors set different speeds of locomotor-like activity through mechanisms acting at various network levels. AMPA and kainate receptors are necessary for generating the highest locomotor frequencies. For coordination, NMDARs are more important than non-NMDARs for conveying the rhythmic signal from the network to the motor neurons during long-lasting and steady locomotor activity. This study reveals that a diversity of ionotropic GluRs tunes the network to perform at different locomotor speeds and provides multiple levels for potential regulation and plasticity.
PMCID: PMC2938926  PMID: 20844601
spinal cord; locomotion; central pattern generator; NMDA; 5HT; AMPA; kainate; NBQX
15.  Synaptic integration of rhythmogenic neurons in the locomotor circuitry: the case of Hb9 interneurons 
Innovative molecular and genetic techniques have recently led to the identification of genetically defined populations of ipsilaterally projecting excitatory interneurons with probable functions in the rhythm-generating kernel of the central pattern generators (CPGs). The role of interneuronal populations in specific motor function is determined by their synaptic inputs, intrinsic properties, and target neurons. In this review we examine whether Hb9-expressing interneurons (Hb9 INs) fulfill a set of criteria that are the hallmarks of rhythm generators in the locomotor circuitry. Induced locomotor-like activity in this distinct population of ventral interneurons is in phase with bursts of motor activity, raising the possibility that they are part of the locomotor generator. To increase our understanding of the integrative function of Hb9 INs in the locomotor CPG, we investigated the cellular mechanisms underlying their rhythmic activity and examined the properties of synaptic inputs from low-threshold afferents and possible synaptic contacts with segmental motoneurons. Our findings suggest that the rhythmogenic Hb9 INs are integral components of the sensorimotor circuitry that regulate locomotor-like activity in the spinal cord.
PMCID: PMC3057624  PMID: 20536922
locomotor-like rhythms; rhythmogenic interneurons; Hb9 interneurons; rhythm-generating kernel; locomotor central pattern generator; Hb9:eGFP transgenic mouse
16.  Activity of Hb9 Interneurons during Fictive Locomotion in Mouse Spinal Cord 
Hb9 interneurons (Hb9 INs) are putative components of the mouse spinal locomotor central pattern generator (CPG) and candidates for the rhythm-generating kernel. Studies in slices and hemisected spinal cords showed that Hb9 INs display TTX-resistant membrane potential oscillations, suggesting a role in rhythm generation. To further investigate the roles of Hb9 INs in the locomotor CPG, we used two-photon calcium imaging in the in vitro isolated whole neonatal mouse spinal cord preparation to record the activity of Hb9 INs, which were subsequently stained for unambiguous genetic identification. We elicited fictive locomotion by transmitter application or by electrically stimulating the caudal tip of the spinal cord. While most Hb9 INs were rhythmically active during fictive locomotion, their activity was sparse and they failed to fire with each cycle of the episode. If Hb9 INs are the principal pacemakers of the CPG in the hemisegment in which they are located, they should direct the firing of motor neurons, with their activity preceding that of their ipsilateral segmental ventral roots. Instead, during each locomotor cycle, onset of Hb9 IN activity lagged behind the onset of the ipsilateral ventral root burst by a mean phase of 0.21 during electrical stimulation and 0.28 during transmitter application. Whole-cell recordings in intact and hemisected spinal cords confirmed the imaging results. Our data suggest that Hb9 INs participate in fictive locomotion, but the delayed onset of activity relative to ipsilateral motoneurons suggests that Hb9 INs are unlikely to be the sole intrasegmental rhythm generating kernel of the CPG.
PMCID: PMC2756057  PMID: 19759307
spinal cord; interneuron; locomotion; central pattern generator; fluorescence microscopy; mice
17.  State-dependent Control of Lumbar Motoneurons by the Hypocretinergic System 
Experimental neurology  2009;221(2):335.
Neurons in the lateral hypothalamus (LH) that synthesize hypocretins (Hcrt-1 and 2) are active during wakefulness and excite lumbar motoneurons. Because hypocretinergic cells also discharge during phasic periods of rapid eye movement (REM) sleep, we sought to examine their action on the activity of motoneurons during this state. Accordingly, cat lumbar motoneurons were intracellularly recorded, under α-chloralose anesthesia, prior to (control) and during the carbachol-induced REM sleep-like atonia (REMc). During control conditions, LH stimulation induced excitatory postsynaptic potentials (composite EPSP) in motoneurons. In contrast, during REMc, identical LH stimulation induced inhibitory PSPs in motoneurons. We then tested the effects of LH stimulation on motoneuron responses following the stimulation of the nucleus reticularis gigantocellularis (NRGc) which is part of a brainstem-spinal cord system that controls motoneuron excitability in a state-dependent manner. LH stimulation facilitated NRGc stimulation-induced composite EPSP during control conditions whereas it enhanced NRGc stimulation-induced IPSPs during REMc. These intriguing data indicate that the LH exerts a state-dependent control of motor activity. As a first step to understand these results, we examined whether hypocretinergic synaptic mechanisms in the spinal cord were state-dependent. We found that the juxtacellular application of Hcrt-1 induced motoneuron excitation during control conditions whereas motoneuron inhibition was enhanced during REMc. These data indicate that the hypocretinergic system acts on motoneurons in a state-dependent manner via spinal synaptic mechanisms. Thus, deficits in Hcrt-1 may cause the coexistence of incongruous motor signs in cataplectic patients, such as motor suppression during wakefulness and movement disorders during REM sleep.
PMCID: PMC2819009  PMID: 19962375
18.  A glycine receptor is involved in the organization of swimming movements in an invertebrate chordate 
BMC Neuroscience  2010;11:6.
Rhythmic motor patterns for locomotion in vertebrates are generated in spinal cord neural networks known as spinal Central Pattern Generators (CPGs). A key element in pattern generation is the role of glycinergic synaptic transmission by interneurons that cross the cord midline and inhibit contralaterally-located excitatory neurons. The glycinergic inhibitory drive permits alternating and precisely timed motor output during locomotion such as walking or swimming. To understand better the evolution of this system we examined the physiology of the neural network controlling swimming in an invertebrate chordate relative of vertebrates, the ascidian larva Ciona intestinalis.
A reduced preparation of the larva consisting of nerve cord and motor ganglion generates alternating swimming movements. Pharmacological and genetic manipulation of glycine receptors shows that they are implicated in the control of these locomotory movements. Morphological molecular techniques and heterologous expression experiments revealed that glycine receptors are inhibitory and are present on both motoneurones and locomotory muscle while putative glycinergic interneurons were identified in the nerve cord by labeling with an anti-glycine antibody.
In Ciona intestinalis, glycine receptors, glycinergic transmission and putative glycinergic interneurons, have a key role in coordinating swimming movements through a simple CPG that is present in the motor ganglion and nerve cord. Thus, the strong association between glycine receptors and vertebrate locomotory networks may now be extended to include the phylum chordata. The results suggest that the basic network for 'spinal-like' locomotion is likely to have existed in the common ancestor of extant chordates some 650 M years ago.
PMCID: PMC2822779  PMID: 20085645
19.  Firing and Cellular Properties of V2a Interneurons in the Rodent Spinal Cord 
The Journal of Neuroscience  2010;30(1):24-37.
Previous studies have shown that a group of ventrally located neurons, designated V2a interneurons, play a key role in maintaining locomotor rhythmicity and in ensuring appropriate left–right alternation during locomotion (Crone et al., 2008, 2009). These V2a interneurons express the transcription factor Chx10. The aim of the present study was to characterize the locomotor-related activity of individual V2a interneurons, their cellular properties, and their detailed anatomical attributes in Chx10-GFP mice. A dorsal horn-removed preparation was developed to allow for visual whole-cell patch recordings from V2a interneurons along the entire lumbar spinal cord while at the same time leaving enough of the spinal cord intact to generate fictive locomotion. During drug-evoked locomotor-like activity, a large proportion of Chx10 cells showed rhythmic firing or membrane potential fluctuations related to either flexor or extensor activity in every lumbar segment. Chx10 cells received predominantly rhythmic excitatory input. Chx10 neurons displayed a wide variety of firing and potential rhythmogenic properties. However, none of these properties was obviously related to the observed rhythmicity during locomotor-like activity. In dual recordings, we found no evidence of Chx10 neuron interconnectivity. Intracellular fills revealed diverse projection patterns with most Chx10 interneurons being local with projections to the central pattern generator and motor neuron regions of the spinal cord and others with long ascending and/or descending branches. These data are compatible with V2a neurons having a role in regulating segmental left–right alternation and ipsilateral motor neuron firing with little effect on rhythm generation.
PMCID: PMC3842458  PMID: 20053884
20.  Flexibility in the Patterning and Control of Axial Locomotor Networks in Lamprey 
In lower vertebrates, locomotor burst generators for axial muscles generally produce unitary bursts that alternate between the two sides of the body. In lamprey, a lower vertebrate, locomotor activity in the axial ventral roots of the isolated spinal cord can exhibit flexibility in the timings of bursts to dorsally-located myotomal muscle fibers versus ventrally-located myotomal muscle fibers. These episodes of decreased synchrony can occur spontaneously, especially in the rostral spinal cord where the propagating body waves of swimming originate. Application of serotonin, an endogenous spinal neurotransmitter known to presynaptically inhibit excitatory synapses in lamprey, can promote decreased synchrony of dorsal–ventral bursting. These observations suggest the possible existence of dorsal and ventral locomotor networks with modifiable coupling strength between them. Intracellular recordings of motoneurons during locomotor activity provide some support for this model. Pairs of motoneurons innervating myotomal muscle fibers of similar ipsilateral dorsoventral location tend to have higher correlations of fast synaptic activity during fictive locomotion than do pairs of motoneurons innervating myotomes of different ipsilateral dorsoventral locations, suggesting their control by different populations of premotor interneurons. Further, these different motoneuron pools receive different patterns of excitatory and inhibitory inputs from individual reticulospinal neurons, conveyed in part by different sets of premotor interneurons. Perhaps, then, the locomotor network of the lamprey is not simply a unitary burst generator on each side of the spinal cord that activates all ipsilateral body muscles simultaneously. Instead, the burst generator on each side may comprise at least two coupled burst generators, one controlling motoneurons innervating dorsal body muscles and one controlling motoneurons innervating ventral body muscles. The coupling strength between these two ipsilateral burst generators may be modifiable and weakening when greater swimming maneuverability is required. Variable coupling of intrasegmental burst generators in the lamprey may be a precursor to the variable coupling of burst generators observed in the control of locomotion in the joints of limbed vertebrates.
PMCID: PMC3223480  PMID: 21743089
21.  Frequency-dependent recruitment of V2a interneurons during fictive locomotion in the mouse spinal cord 
Nature communications  2011;2:274.
The principles governing the recruitment of interneurons during acceleration in vertebrate locomotion are not known. In the mouse, the V2a spinal interneurons are dispensable for left-right coordination at low locomotor frequencies, but their function is essential for maintaining left-right coordination at high frequencies. Here we explore the mechanisms driving this frequency-dependent role, using four methods to determine how V2a interneurons are recruited at different locomotor frequencies. We show that half the V2a interneurons receive rhythmic locomotor synaptic drive which increases with cycle frequency, recruiting more of the neurons to fire at higher frequencies. The other V2a interneurons do not receive locomotion-related synaptic drive, and are not recruited into the locomotor network at any frequency. The increased role of V2a interneurons at higher locomotor frequencies arises from increased synaptic drive to recruit subthreshold oscillating V2a neurons, and not from recruitment of a second set of silent V2a interneurons.
PMCID: PMC3597081  PMID: 21505430
22.  Recovery of neuronal and network excitability after spinal cord injury and implications for spasticity 
The state of areflexia and muscle weakness that immediately follows a spinal cord injury (SCI) is gradually replaced by the recovery of neuronal and network excitability, leading to both improvements in residual motor function and the development of spasticity. In this review we summarize recent animal and human studies that describe how motoneurons and their activation by sensory pathways become hyperexcitable to compensate for the reduction of functional activation of the spinal cord and the eventual impact on the muscle. Specifically, decreases in the inhibitory control of sensory transmission and increases in intrinsic motoneuron excitability are described. We present the idea that replacing lost patterned activation of the spinal cord by activating synaptic inputs via assisted movements, pharmacology or electrical stimulation may help to recover lost spinal inhibition. This may lead to a reduction of uncontrolled activation of the spinal cord and thus, improve its controlled activation by synaptic inputs to ultimately normalize circuit function. Increasing the excitation of the spinal cord with spared descending and/or peripheral inputs by facilitating movement, instead of suppressing it pharmacologically, may provide the best avenue to improve residual motor function and manage spasticity after SCI.
PMCID: PMC4026713  PMID: 24860447
serotonin; noradrenaline; motoneuron; persistent inward currents; reflexes; spinal cord injuries
23.  The Spinobulbar System in Lamprey 
Brain research reviews  2007;57(1):37-45.
Locomotor networks in the spinal cord are controlled by descending systems which in turn receive feedback signals from ascending systems about the state of the locomotor networks. In lamprey, the ascending system consists of spinobulbar neurons which convey spinal network signals to the two descending systems, the reticulospinal and vestibulospinal neurons. Previous studies showed that spinobulbar neurons consist of both ipsilaterally- and contralaterally-projecting cells distributed at all rostrocaudal levels of the spinal cord, though most numerous near the obex. The axons of spinobulbar neurons ascend in the ventrolateral spinal cord and brainstem to the caudal mesencephalon and within the dendritic arbors of reticulospinal and vestibulospinal neurons. Compared to mammals, the ascending system in lampreys is more direct, consisting of excitatory and inhibitory monosynaptic inputs from spinobulbar neurons to reticulospinal neurons. The spinobulbar neurons are rhythmically active during fictive locomotion, representing a wide range of timing relationships with nearby ventral root bursts including those in phase, out of phase, and active during burst transitions between opposite ventral roots. The spinobulbar neurons are not simply relay cells because they can have mutual synaptic interactions with their reticulospinal neuron targets and they can have synaptic outputs to other spinal neurons. Spinobulbar neurons not only receive locomotor inputs but also receive direct inputs from primary mechanosensory neurons. Due to the relative simplicity of the lamprey nervous system and motor control system, the spinobulbar neurons and their interactions with reticulospinal neurons may be advantageous for investigating the general organization of ascending systems in the vertebrate.
PMCID: PMC2246055  PMID: 17716741
lamprey; spinal cord; locomotion; spinobulbar; reticulospinal
24.  Spinal interneurons differentiate sequentially from those driving the fastest swimming movements in larval zebrafish to those driving the slowest ones 
Studies of neuronal networks have revealed few general principles that link patterns of development with later functional roles. While investigating the neural control of movements, we recently discovered a topographic map in the spinal cord of larval zebrafish that relates the position of motoneurons and interneurons to their order of recruitment during swimming. Here, we show that the map reflects an orderly pattern of differentiation of neurons driving different movements. First, we use high-speed filming to show that large amplitude swimming movements with bending along much of the body appear first, with smaller, regional swimming movements emerging later. Next, using whole-cell patch recordings, we demonstrate that the excitatory circuits that drive large amplitude, fast swimming movements at larval stages are present and functional early on in embryos. Finally, we systematically assess the orderly emergence of spinal circuits according to swimming speed using transgenic fish expressing the photoconvertible protein, Kaede, to track neuronal differentiation in vivo. We conclude that a simple principle governs the development of spinal networks in which the neurons driving the fastest, most powerful swimming in larvae develop first with ones that drive increasingly weaker and slower larval movements layered on over time. Because the neurons are arranged by time of differentiation in the spinal cord, the result is a topographic map that represents the speed/strength of movements at which neurons are recruited and the temporal emergence of networks. This pattern may represent a general feature of neuronal network development throughout the brain and spinal cord.
PMCID: PMC2796107  PMID: 19864569
Spinal cord; Development; Networks; Locomotion; Swimming; Central Pattern Generator
25.  Nanomolar Oxytocin Synergizes with Weak Electrical Afferent Stimulation to Activate the Locomotor CPG of the Rat Spinal Cord In Vitro 
PLoS ONE  2014;9(3):e92967.
Synergizing the effect of afferent fibre stimulation with pharmacological interventions is a desirable goal to trigger spinal locomotor activity, especially after injury. Thus, to better understand the mechanisms to optimize this process, we studied the role of the neuropeptide oxytocin (previously shown to stimulate locomotor networks) on network and motoneuron properties using the isolated neonatal rat spinal cord. On motoneurons oxytocin (1 nM–1 μM) generated sporadic bursts with superimposed firing and dose-dependent depolarization. No desensitization was observed despite repeated applications. Tetrodotoxin completely blocked the effects of oxytocin, demonstrating the network origin of the responses. Recording motoneuron pool activity from lumbar ventral roots showed oxytocin mediated depolarization with synchronous bursts, and depression of reflex responses in a stimulus and peptide-concentration dependent fashion. Disinhibited bursting caused by strychnine and bicuculline was accelerated by oxytocin whose action was blocked by the oxytocin antagonist atosiban. Fictive locomotion appeared when subthreshold concentrations of NMDA plus 5HT were coapplied with oxytocin, an effect prevented after 24 h incubation with the inhibitor of 5HT synthesis, PCPA. When fictive locomotion was fully manifested, oxytocin did not change periodicity, although cycle amplitude became smaller. A novel protocol of electrical stimulation based on noisy waveforms and applied to one dorsal root evoked stereotypic fictive locomotion. Whenever the stimulus intensity was subthreshold, low doses of oxytocin triggered fictive locomotion although oxytocin per se did not affect primary afferent depolarization evoked by dorsal root pulses. Among the several functional targets for the action of oxytocin at lumbar spinal cord level, the present results highlight how small concentrations of this peptide could bring spinal networks to threshold for fictive locomotion in combination with other protocols, and delineate the use of oxytocin to strengthen the efficiency of electrical stimulation to activate locomotor circuits.
PMCID: PMC3962494  PMID: 24658101

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