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Circuits controlling vertebrate
locomotion: moving in a new direction
Martyn Goulding

Abstract | Neurobiologists have long sought to understand how circuits in the nervous
system are organized to generate the precise neural outputs that underlie particularbehaviours. The motor circuits in the spinal cord that control locomotion, commonlyreferred to as central pattern generator networks, provide an experimentally tractablemodel system for investigating how moderately complex ensembles of neurons generate
select motor behaviours. The advent of novel molecular and genetic techniques coupledwith recent advances in our knowledge of spinal cord development means that acomprehensive understanding of how the motor circuitry is organized and operates may be
within our grasp.
Central pattern generator(CPG). A network of neurons
that autonomously generates
rhythmic patterns of activity.

Molecular Neurobiology
Laboratory, The Salk Institutefor Biological Studies,
10010 North Torrey Pines
Road, La Jolla, California
92037, USA.
e-mail: goulding@salk.edu
doi:10.1038/nrn2608

Motor tasks are key components of the behaviouralrepertoire of all animals1–3. Although animals typicallyexhibit quite varied and complex patterns of motor activity, many of their simpler motor behaviours — including
breathing, chewing, peristalsis, swimming, scratching andwalking — are well suited to experimental analysis4–13.
The analysis of motor behaviours has long been at
the centre of efforts to understand how the nervous
system is organized and functions, with Sherrington’s
pioneering studies providing important insights into
the integrative nature of neural pathways, the reflex arc
and the control of reciprocal motor actions by central
inhibitory pathways14. Subsequent studies in the cat, by
Eccles, Lundberg, Jankowska and colleagues went a long
way toward defining the spinal reflex circuitry, including the properties of the constituent interneurons andtheir actions on motor neurons15–18. The idea, favoured
by Sherrington, that complex motor behaviours, including locomotion, were generated by chains of reflex
actions14, was countered by Brown, who provided evidence that intrinsic networks in the spinal cord cangenerate rhythmic locomotor-like patterns of activity 19.
This observation gave rise to the concept of the central
pattern generator (CPG), a neuronal network that is capable of generating an organized pattern of motor activity
independently of sensory inputs and which was firstdescribed in invertebrates20. In the vertebrate spinal cord,such networks function as local “control and command”
centres5,6,13,21 to generate rhythmic axial and limb movements. Descending inputs from the brainstem, basal
ganglia and cortex control the selection and shaping of
outputs from the locomotor CPG, with further layers

nature reviews | Neuroscienceof modulation coming from sensory and vestibularpathways that converge on CPG neurons22–25 (FIG. 1).
The vertebrate locomotor CPG comprises a distributed
network of interneurons and motor neurons, which upon
appropriate stimulation generates an organized motor
rhythm that replicates the patterns of motor activity seen
during repetitive locomotor tasks such as walking andswimming (FIG. 2). The central organizing feature of the
motor circuitry is the grouping of motor neurons into discrete operational units, called motor pools, each of which
innervates a single muscle14. The graded recruitment andactivation of motor neurons within a pool26–28 underlies
the variable changes in muscle tension that are necessary
for smooth muscle movement and postural control. Motorneurons integrate a range of convergent inputs, although
it is likely that much of the integration that gener­ates
coordinated motor activity takes place upstream in thelocomotor interneuron network. Fast synaptic and slower
modulatory interactions between locomotor interneurons sculpt the patterns of motor neuron activity thatcoordinate limb and body movements; however, theseinteractions remain largely undefined.
Past efforts to probe the neuronal networks responsible
for generating motor behaviours have typically relied onclassical systems neuroscience approaches, such as combinations of neuronal recordings and pharmaco­logicalmanipulations. Although these studies have provided
important insights, they are limited by difficulties in
reproducibly identifying and manipulating the neurons
within these networks. In recent years, the convergence
between developmental genetics and physiological andbehavioural systems approaches has opened up new
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REVIEWS
avenues for identifying and manipulating specific neuronal cell types. In this Review, I will highlight how efforts
to understand the genetic regulation of neuronal specification in the embryonic spinal cord are being mergedwith systems approaches to study the organization of the
spinal locomotor network in vertebrates29–38. In particular, the increased sophistication of genetic manipulations
in mice and other model vertebrate organisms such aszebrafish39–41 is facilitating a more precise dissectionof the motor circuitry and the contribution different
neuronal cell types make to motor behaviours.

Lessons from other rhythmic model systems
The cellular mechanisms that underlie the rhythmic
motor behaviours produced by CPG networks havebeen explored in a number of contexts, including insect
flight, swimming in the mollusc Clione, gut movements in crustaceans, and swimming and respirationaCortexSpinal cord
(locomotion,
spinal reflexes)

Cerebellum

Basal ganglia
Hypothalamus
Brainstem(respiration, chewing, eye movements)b

Forebrain

Midbrain

Hindbrain

Spinal cord

Motor cortex
Cerebellum

Thalamus

MLR

Basal ganglia

Pons/medulla
RtS pathway
VS pathway
RbS pathway

Locomotor
CPG network
Proprioceptive
sensory
feedback
MuscleFigure 1 | Organization of the locomotor system in vertebrates. a | Schematic of the
Reviews | Neurosciencerodent CNS showing the neural structures that make up theNature
motor pathways
controlling
simple behaviours such as mastication, respiration and locomotion. b | Motor pathways
in aquatic and terrestrial vertebrates share a similar neuroanatomical structure. Local
control of muscle movements is regulated by pools of motor neurons in the spinal cordthat are part of a dispersed locomotor central pattern generator (CPG) network. Spinal
motor centres are modulated by proprioceptive sensory feedback through sensory
afferents. Descending reticulospinal (RtS), rubrospinal (RbS) and vestibulospinal (VS)pathways control the locomotor network in the spinal cord, although the reticulospinal
pathway is the primary pathway for initiating locomotion. The reticulospinal pathway
can be activated by the mesencephalic locomotor region (MLR), which has inputs fromthe basal ganglia and the thalamus. The cerebellum coordinates motor behaviours by
mediating sensory and internal feedback and optimizing the motor pattern to the task
at hand. It also coordinates spinal motor actions via supraspinal motor pathways.
Connections from the motor cortex refine and initiate motor actions (dotted arrow).
The black arrows indicate direct command pathways, the grey arrows indicate
feedback pathways. Part a is modified, with permission, from REF. 5  (2003) Macmillan
Publishers Ltd. All rights reserved.
508 | july 2009 | Volume 10

in vertebrates3–6,11,12. The relative simplicity of thesesystems, best exemplified by the crustacean stomatogastric nervous system (STG), have facilitated efforts
to define how rhythmic networks are organized andoperate4, and have highlighted the central role of CPG
neurons in rhythm generation (BOX 1). Moreover, it is
now apparent that the properties of CPG neurons arestrongly influenced by neuromodulators4,6,42. Many CPG
networks are regulated by multiple neuromodulatorsthat regulate neuronal excitability and synaptic transmission in a cell-specific and state-dependent manner.
As such, these neuromodulators have important roles
in configuring the CPG output. Another key feature ofthese networks is the differential recruitment of CPG
neurons during various motor behaviours. This is seenin the crustacean STG43,44, in the leech locomotor network45,46 and in the turtle spinal cord47, in which particular interneurons are active during both swimmingand scratching while other interneurons are selectively
recruited for each task. However, it is not clear whether
the CPG is reconfigured in a task-dependent manner orwhether motor systems are comprised of multiple CPGs
that use varying combinations of neurons from within
the motor network for different behaviours.
Studies of simpler CPG networks have defined two
general mechanisms for rhythm generation: pacemakerdriven systems and mechanisms that rely on the properties of the constituent neurons. The pyloric rhythmin the STG is largely driven by pacemaker neurons48,49,
and neurons with pacemaker properties have been
identified in the pre-Botzinger complex that generates
the inspiratory rhythm50. In other systems, oscillatory
motor activity seems to be an emergent property that
requires synaptic coupling between neurons4,5. Simple
examples of the latter are the half-centre oscillator thatdrives swimming in Clione 3,51 and the beating of theheart in leech42,46. These oscillators depend on reciprocal inhibitory interactions between neurons, which arealso thought to be important for the swimming CPG
rhythm5,7,52,53. Nonetheless, the observation that rhythmic activity continues in the rodent spinal cord when
all fast inhibitory transmission is blocked54 is but one
indication of the rudimentary understanding we have of
the cellular mechanisms that underlie locomotor rhythm
generation in the vertebrate cord.

Insights from the swimming CPG
The lamprey and amphibian spinal cord have provided
key insights into the structure of the swimming CPG andthe synaptic interactions that produce swimming movements5–8. Although the swimming movements of aquatic
vertebrates differ markedly from the limb-dependent
motor behaviours seen in terrestrial vertebrates, the overall organization and neuronal makeup of the locomotor
system is remarkably conserved5–8,13. Because of this, theorganization of the swimming motor system is likely to
be useful for understanding the network structure of thelocomotor CPG in terrestrial vertebrates that use theirlimbs to move. Studies in the lamprey have revealed
four general classes of neurons as core elements of theswimming CPG5. These have functional and anatomical
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REVIEWSa Swimming CPG (lamprey)b Walking CPG (mouse, cat)

Intact animal

Intact animal

L-5

R-Tr

R-5

L-Tr

R-25

R-VI

R-46

L-VI
0.1 s

Isolated spinal cord
R-6

0.2 s
Isolated spinal cord

R-26

R-L2

R-L5

L-L2

L-L5

R-L2

R-6
R-26

L-L2

R-46
1s

R-L5
2s

Figure 2 | Rhythmic motor patterns underlying vertebrate locomotion.
Nature Reviews
a | Examples of spinal motor activity during swimming in the lamprey.
The top| Neuroscience
traces show
electromyograph (EMG) recordings of different myotomes located at different axial levelsin the intact animal. The bottom traces show ventral root recordings from the isolated
spinal cord that exhibit a slow pattern of rhythmic motor activity. The motor outputs of theintact animal and isolated spinal cord show the same patterns of motor coordination andsegmental lag. b | Walking motor behaviour. The top traces are EMG recordings, showing
muscle activity in the cat hindlimb. The bottom traces show the results from an isolated
spinal cord preparation from a postnatal day 0 mouse. These are electroneurogram (ENG)
recordings from L2 and L5 ventral roots following the induction of fictive walking by
NMDA (N-methyl-d-aspartate) and serotonin (5‑HT). The ENG traces measureflexor-related (L2) and extensor-related (L5) motor activity. Figure is modified, withpermission, from Ref. 3  (1999) Oxford University Press.

equivalents in the frog tadpole and in zebrafish, alongwith developmental, and presumably functional,
homologues in birds and mammals (BOXES 2,3).

Commissural neurons
Neurons whose axons crossfrom one side of the spinal
cord to the other.

Locomotor circuits in terrestrial vertebrates.
The transition from water to land-based locomotion
during vertebrate evolution resulted in a marked change
in the mode by which vertebrate animals move. Althoughsideways flexion of the torso provides an excellent mechanism for movement through water, this form of propulsion is largely ineffective on land or in air. Consequently,
most terrestrial vertebrates, besides snakes, primarily
use their limbs for propulsion, with trunk movements
often augmenting the gait by amplifying and prolonging movements. Weight bearing, postural changes andvariations in limb placement also come into play withnature reviews | Neuroscienceland-based locomotion, and as a result the spinal
circuitry required for limb-driven locomotion is more
complex than that needed for swimming. One indicationof this is the multiple spinal interneuron cell types thatare known to contribute to the sensory reflex-dependent
modulation of motor outputs in the cat 15–18. This suggeststhat much of the neural diversity seen in the spinal cordsof terrestrial vertebrates may result from the increased
level of sensory information that is used to inform andmodulate the spinal motor machinery.
To date most of the studies examining the neuralcircuits involved in quadripedal locomotion have been
carried out in cats and rodents2–3,15–18,25,32–34. Experimentsin the cat have been key to understanding how spinal
reflex pathways are organized and the role that sensory
feedback has in shaping movements. Three principles
have emerged from analyses in the cat. First, sensory
inputs are important for initiating and correcting the
locomotor rhythm. Second, sensory inputs change
the amplitude of the motor output and regulate phasechanges during stepping. Third, the transmission ofreflexes and their actions vary with the step phase.
Recordings from the cat spinal cord have also identified and characterized some of the interneuron cell
types that are involved in these spinal sensorimotor
pathways15–18. More recent efforts to dissect the walking CPG have relied heavily on the isolated neonatal rat
and mouse spinal cord preparation13,32,36,39,40,55–57. Eachmodel system provides complementary information
about the makeup and organization of the locomotor
circuitry. Consequently, the value of merging these tworather disparate perspectives into a singular comprehensive view of the spinal locomotor circuitry cannot be
underestimated. For example, the identification in miceof cell types previously characterized in the cat enables
direct comparisons between the two systems. Geneticfate mapping of Renshaw cells and systems analysis ofreciprocal inhibitory pathways in the mouse mark the
beginning of such efforts58–60.

The mammalian locomotor CPG
The spinal CPG in walking mammals is a distributed
network with centres at cervical and lumbar levels that
control forelimbs and hindlimbs, respectively 2,3. The
hindlimb CPG has been studied extensively, with earlyexperiments in the cat showing that the lumbar andsacral spinal cord can elicit a normal pattern of walking
activity when isolated from the rest of the CNS and fromsensory inputs19,21. Smaller regions of the spinal cord canalso generate coordinated motor activity. In the cat, thethree segments from L6 to S1 retain the ability to generate a normal pattern of motor activity for ankle extensor
and flexor muscles21. This and other studies indicate that
CPG network for each limb comprises multiple interconnected modules that control the movement of eachjoint. Commissural neurons and propriospinal connections
secure coordination between both sides of the spinal cordand between the forelimbs and hindlimbs. CPG activity isalso coupled across multiple joints for each limb.
The spatial arrangement of components of the hindlimb locomotor CPG network has been mapped in theVolume 10 | july 2009 | 509

REVIEWS
Ventricular zone
Innermost layer of theembryonic spinal cord that
contains dividing progenitor
cells.

Alar plate
Dorsal region of the ventricularzone in the embryonic spinal
cord.

rodent lumbar cord61–63. Evidence that the core CPG
components are located in the ventral half of the spinal
cord63 is supported by studies showing that activitydependent markers are upregulated in lamina VII
and lamina VIII during locomotor-related tasks 64–67.
Moreover, many ventrally-derived embryonic cell types
with demonstrated roles in locomotion populate lamina
VII and VIII in the adult spinal cord29,33,40,67, as do dI6
commissural neurons that arise in the dorsal half of theneural tube68,69 (FIG. 3).

Molecular identification of locomotor interneurons
The elucidation of the genetic programmes that control
neuronal patterning in the embryonic spinal cord haveenabled researchers to molecularly identify and genetically manipulate locomotor interneurons in mice.
Underpinning these studies was the finding that neuronal identity in the spinal cord is primarily determinedby the dual activities of two morphogen gradients that
impart dorsoventral positional information to dividing
neural progenitors in the ventricular zone29,70. Ventrally,
the notochord and floor plate produce Sonic hedgehog(Shh)29,30, while dorsally, the epidermis over­lying theneural tube and roof plate secretes bone morpho­genetic
proteins (BMPs)70. The opposing activities of Shh andthe dorsally derived BMPs restrict the expression of patterning factors to spatially limited subsets of ventricularzone progenitors (FIG. 3). This results in the ventricular zone
being subdivided into discrete dorsoventral progenitor domains (eleven in mouse and chick) that generate
different classes of embryonic neurons31.
In the mouse dorsal alar plate, six progenitor domains
generate early-born dI1-dI6 neurons, as well as two lateborn classes of dorsal interneurons31,68,69. In the ventral
half of the neural tube, five classes of neurons — the‘generic’ motor neurons and four classes of putative ‘core’
CPG interneurons (the so-called V0, V1, V2 and V3
neurons29–31,40,71–77) are produced. Each of the spinal CPG
interneuron classes exhibits a unique phenotype. V0 neurons are commissural neurons that extend axons rostrally
for 2–4 spinal cord segments in the embryonic cord74,75.
By contrast, most V3 neurons are excitatory commissural
neurons that extend a caudally projecting primary axon40
Box 1 | Properties of neurons that contribute to rhythm generation
Neurons within a rhythmic circuit typically exhibit one or more of the following properties:• Endogenous bursting. When isolated, some neurons fire in bursts spontaneously or in
response to neuromodulators, thus functioning as pacemaker neurons11,49.
• Postinhibitory rebound. When a neuron is hyperpolarized, the membrane potential
reverses producing either a single action potential or a train of action potentials.
Neurons that exhibit these properties in a network can contribute to rhythm generation49.
• Spike frequency adaptation. These neurons will initially fire action potentials, but
subsequently accommodate and cease firing.
• Plateau potentials. These occur when transient excitatory inputs lead to long-lived
depolarized states or transient inhibition hyperpolarizes the cell. Plateau potentials cancontribute to bistability in neurons and oscillatory network activity. Interneurons andmotor neurons in many motor systems exhibit plateau potentials131–133.
The cellular, synaptic and modulatory mechanisms regulating central patterngenerator activity are discussed in Refs 4,5.

510 | july 2009 | Volume 10(M.G., unpublished observations). The V1 neurons, liketheir Xenopus and zebrafish homologues, are inhibitory
interneurons76–80 with axons that project ipsilaterally androstrally 76,80. The V2 neurons, which comprise a mixed
population of glutamatergic V2a neurons and inhibitory
V2b neurons71–73, also project ipsilaterally, but preliminary studies suggest that they extend their axons caudally
across multiple spinal cord segments (M.G, unpublishedobservations). In addition to the V interneuron classes,
dorsally derived dI6 neurons are also likely to contributeto the spinal locomotor CPG67–69.
Each neuron class seems to comprise multiple
neuronal cell types that share a number of common anatomical features. For example, newborn motor neurons
can differentiate into either visceral or somatic motor
neurons, with the latter acquiring distinct columnar andpool identities29,30. The V1 class is also diverse, comprising two known types of local circuit inhibitory neuron(Renshaw cells and Ia inhibitory interneurons58.59) and
one or more undefined inhibitory neuron subtypes.
The V2, V3 and dI6 populations are also made up ofmultiple molecularly distinct cell types40,71 (T. Hendricks
and M.G, unpublished observations), with each subtype
likely to have specialized roles in locomotor control.

Commissural interneurons: both halves in stepExperiments in rodents and cats have demonstrated theimportance of commissural connections for coordinatedleft–right limb movements81,82. Moreover, blocking fast
inhibitory transmission results in the loss of left–rightalternation and the production of a synchronous patternof left–right motor activity in the isolated spinal cord54.
Thus, inhibitory commissural pathways have criticalroles in controlling left–right alternation during walking, much as they do in swimming 5–8,52,53,83,84. V0 neurons
are one of two classes of molecularly-defined inhibitory
interneuron found in lamina VIII67. These commissural neurons are derived from progenitors that express
the homeodomain transcription factor Dbx1, which is
required for their development and commissural connectivity 75. V0 neurons are necessary for proper couplingof the left and right hindlimb CPGs during walking67, as
isolated spinal cords from Dbx1–/– mice exhibited intermittent periods of synchronous hopping-like activity.
However, as periods of normal alternation still occur in
the Dbx1 mutant cord, the V0 neurons cannot be solely
responsible for securing left–right alternation67. The dI6
neurons, which are also inhibitory commissural neurons(T. Hendricks and M.G, unpublished observations), may
contribute to the crossed inhibitory pathways that secureleft–right alternation as well.
Mice lacking the receptor tyrosine kinase Epha4, its
ligand ephrin B3, or the Nck adaptor protein also exhibit
a loss of left–right alternation85–87, with experiments in
the isolated spinal cord pointing to an intrinsic spinal
cord defect 86. Because Epha4 is expressed in many
spinal-cord cell types, it has been difficult to define withany precision the changes in connectivity that underlie the hopping gait of these mice. Interestingly, manyof the Epha4 expressing neurons in the cord are excitatory neurons that project ipsilaterally 88. Eph-dependent
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Box 2 | The four primary locomotor cell types found in the lamprey cord
Four functional classes of neurons make up the swimming central pattern generator(CPG) in lamprey (see the figure):• Segmentally organized motor neurons (MNs) that innervate each adjacent axial
myotome.
• Glycinergic commissural interneurons (CINs) project to the opposite side of the spinal
cord. During swimming, inhibitory connections provide the mid-cycle inhibition that
ensures that the axial muscles on each side of the body contract out of phase with thoseon the opposite side.
• Ipsilaterally-projecting inhibitory L‑interneurons (IINs) that provide inhibition to motor
neurons and to CINs. Their exact role in swimming has not been defined.
• Excitatory glutamatergic neurons (EINs) that project to all three other CPG neuron cell
types. These cells, or a proportion of these cells, are rhythmically active and provide
rhythmic drive to motor neurons and other CPG neurons during swimming. Excitatorycommissural neurons are also present in the lamprey cord134, however their function is
not known.
Swimming CPG(lamprey, Xenopus, zebrafish) Descending reticulospinal inputs

Axial
myotome

EIN

CIN

CIN

EIN

MN

IIN

IIN

MN

Left

Lineage tracing studies
Techniques that allow theprogeny of a cell in the embryo
to be traced.

Axial
myotomeRight| Neurosciencesignalling has an importantNaturerole inReviewsaxon guidance,
and ithas been suggested that ipsilateral excitatory CPG neurons may either aberrantly cross the midline or makeectopic connections with commissural neurons in the
Epha4 mutant. Recently, it has been shown that V2a neurons are ipsi­lateral components of a commissural pathwaythat secures left–right alternation39. As some V2a neurons
express Epha4 (Ref. 72), defects in V2a connectivity might
contribute to the Epha4/ephrin B3/Nck phenotype.
Genetic approaches in zebrafish are now beginningto shed light on the commissural pathways that control
swimming movements, which were previously investigated using pharmacological manipulations52,53,84,85.
Mutations in the glycine receptor b2 gene (glrb2) thatprevent glycine receptor clustering cause trunk muscles
on both sides of the animal to contract synchronously 89.
Likewise, fish lacking GlyT1 glycine transporter activity
have enhanced glycinergic transmission and are unable
to generate rhythmic swimming movements90. Althoughthese findings support a role for glycinergic commissural
neurons in coupling the CPG networks in each half ofthe cord, both mutations affect inhibitory transmission nonspecifically. To date it has not been possible toselectively inactivate inhibitory commissural neurons inzebrafish. Moreover, there are likely to be multiple glycinergic commissural neurons in swimming vertebrates:
the commissural connections typically depicted in schematics of the swimming CPG (see ref. 5) probably derivefrom more than one cell type.
Excitatory neurons are also components of the locomotor commissural network, with the V3 neurons being
the major class of excitatory commissural neurons in thenature reviews | Neurosciencemouse spinal cord40. V3 neurons have an important rolein establishing a stable and balanced locomotor rhythm.
Although largely dispensable for left–right alternation,
they are important for the production of a symmetrical
motor output from the spinal cord40. The homologuesof V3 neurons in fish have not been identified; however,
VeMe (ventromedial) and UCoD (unipolar commissuraldescending) cells show marked similarities in their morphology and neurotransmitter phenotypes91,92 (FIG. 4). A
class of excitatory commissural neurons in the zebrafish
hindbrain, the spiral fibre neurons, can also regulate
motor behaviours through descending reticulospinal
pathways. The spiral fibre neurons form a circuit that
controls fast turning movements and the escape reflex. In
space cadet mutant fish, spiral fibre axons no longer crossthe ventral commissure to innervate Mauthner cells andinhibitory PHP interneurons, which are presynaptic to
Mauthner cells93. This leads to abnormal turning whenthe escape reflex is activated.

Ipsilateral interneurons: the story so far
Three major populations and one minor population ofgenetically defined interneurons in the embryonic andneonate mouse spinal cord are likely to be core constituents of the locomotor CPG. The V1 and V2b neurons
generate inhibitory cell types, whereas the V2a andHb9-expressing neurons are excitatory. The V1-derived
neurons are well characterized, with lineage tracing studies showing that Renshaw cells and Ia inhibitory inter­neurons are derived from this population58,59. However,these comprise less than 25% of the V1-derived cells,
demonstrating that there are additional uncharacterized
V1-derived cell types in the adult. Although molecularmarkers that are selectively expressed in non-Renshaw,
non-Ia inhibitory V1 neurons have been found (M.G.,
unpublished observations), the neurons expressing thesemarkers have not been correlated with any of the functional cell types previously identified in the cat, such as
Ib inhibitory neurons.
The Gata2/3-expressing V2b interneurons areinterspersed with V1 neurons in lamina VII71–73,94. These
neurons differ from their V1 counterparts in that theirprimary axons project caudally (M.G., unpublished observations). Although the adult progeny of V2b neurons havenot been identified, these cells might contribute to reciprocal inhibitory pathways, as disynaptic inhibition is stillpresent in mice that lack V1 neurons60. The V2b cells are
one likely source of these inhibitory connections, raising the intriguing possibility that certain physiologicallydefined classes of spinal interneurons are more hetero­geneous than previously thought and may be derived
from more than one embryonic class of interneuron.
Less is known about the ipsilaterally-projecting
excitatory interneurons in the rodent spinal cord. Of thethree molecularly defined classes that have been identified in ventral motor regions, only the Hb9 neurons
have been characterized in detail 95–98. Hb9+/VGlut2+
neurons are located medially in lamina VIII at lower
thoracic-upper lumbar levels of the spinal cord. They area small group of neurons, whose embryonic origin is notknown as most Hb9 cells in the spinal cord differentiate
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Fictive locomotion
Locomotion that is initiated in
the absence of sensory
feedback and descending
control from the cortex.

as motor neurons. Glutamatergic Hb9 neurons exhibit
a number of cellular properties that suggest a role inrhythm gener­ation, including rhythmic oscillations
in membrane potential and pronounced post-inhibitory
rebound when hyperpolarized95. Unfortunately, genetic
tests to determine whether the Hb9 interneurons havea role in rhythm generation have been hampered by theexpression of Hb9 in motor neurons and other spinal
interneuron cell types.
A growing arsenal of genetic tools has facilitatedinvestigation of the contribution that these geneticallydefined interneurons make to the locomotor CPG. Two
ipsiltateral CPG interneuron populations have been
analysed so far. V2a neurons were ablated in the mouseby selectively expressing the diptheria-toxin A subunit(DTA) in Chx10-expressing neurons39. This resulted in apartial uncoupling of the left and right halves of the spinal

Box 3 | Putative phylogenetic relationship between spinal cord neurons
The table below illustrates what we know about the relationship between neurons
identified in the spinal cords of different species. aINs and CiA neurons seem to beequivalent to the inhibitory L‑ interneurons of the lamprey and V1 neurons in themouse21,58,59,79,80. In Xenopus, the dIN glutamatergic neurons seem to be the major sourceof ipsilateral excitatory input in the hindbrain and ventral spinal cord7,135–136. dIN neurons
might be homologous to CiD neurons in zebrafish, which express the Chx10transcription factor103. CiD neurons, are rhythmically active during fictive swimming andprovide the main source of on-cycle excitation to the swimming central patterngenerator 103,121,122. Glycinergic inhibitory commissural interneurons have an essential
role in generating these alternating outputs between each half of the spinal
cord5,52–54,83,84. In Xenopus, the commissural interneurons (CINs) that mediate reciprocal
inhibition have been characterized in some detail83,84. They typically fire in phase with
ipsilateral motor neurons once per swimming cycle. There are multiple anatomically,
distinct populations of CINs, including CoPA, CoSA, CoLA, UCoD and CoBL cells in thezebrafish spinal cord91,92,122. Although these cells are largely characterized anatomicallyand to a lesser extent molecularly, their functions in locomotion have not been
described. CoBL and Evx2+ MCoD neurons are both active during swimming122. CoBL
cells are bifurcating glycinergic neurons. The excitatory commissural MCoD neurons are
preferentially recruited during slow swimming movements. They seem to be necessary
for slow swimming, but are dispensable for coordinating the left–right alternation ofsegmental motor neurons during fast swimming movements122. The inhibitory CINs,
excitatory interneurons (EINs) and L‑interneurons in the lamprey have not beenmolecularly characterized. The zebrafish homologues of V3 neurons might be VeMe and
UCoD cells, but they are yet to be identified. UCoD neurons are similar to commissural
V3 interneurons (glutamatergic with descending axons).

Jawless fish
(lamprey)

Bony fish(zebrafish)

Amphibian(Xenopus)

Mammal (mouse)

Inhibitory CINs

CoBL neurons

CINs

dI6 neurons (Lbx1+)

CoSA neurons
Excitatory CINs
EINs

V0D neurons (Dbx1+,
Evx1/2–)MCoD neurons

Undetermined

V0V neurons (Evx1/2+)

UCoD neurons

Undetermined

V3v neurons (Sim1+)

CiD neurons

dINs

V2a neurons (Chx10+)

VeMe neurons*

V3 neurons (Sim1+)
Hb9+ neurons

L-interneurons

CiA neurons
eLD neurons

aINs

V1 neurons (En1+)V2b neurons (Gata3+)* VeMe cells also have descending axons that may cross the ventral midline and are
excitatory.

512 | july 2009 | Volume 10

cord. The resultant loss of left–right alternating activity
indicates that the V2a neurons are ipsilateral components of a spinal commissural pathway that coordinates
left–right hindlimb movements in mice. It is likely that
the V2a neurons serve this function by providing excitatory drive to spinal commissural neurons, as V2 axon
terminals contact a subset of V0 commissural neurons39.
Interestingly, the selective ablation of the V2a neurons
also results in increased variability of the step cycle period
and amplitude of the loco­motor rhythm, suggesting thesecells may also contribute to rhythm generation39.
The function of the V1 neuron population in
shaping motor outputs in the isolated spinal cord has
been analysed with three complementary genetic
approaches36. The first used mice lacking the Pax6 gene,as these animals exhibit developmental defects that leadto the selective loss of V1 neurons in the neonate spinal
cord. In the second approach V1 neurons were ablated bycrossing mice expressing Cre recombinase under controlof the engrailed 1 (En1) promoter with a mouse that conditionally expresses DTA, thereby driving the selective
expression of DTA in V1 neurons. The third approach,also using the Cre-loxP system, involved conditionallyexpressing the receptor for allatostatin99, an insect neuro­
peptide, in V1 neurons (FIG. 5). Using the latter method
V1 neurons were acutely inactivated, thereby circumventing any compensatory changes that might occur during
development. With all three approaches, the inactivation
or deletion of the V1 neurons resulted in a marked prolongation of the step cycle36. Furthermore, preliminary
results demonstrate that blocking neurotransmission in
the V1 neurons following the expression of tetanus toxin(TeNT) also slows the motor rhythm (M.G, unpublishedobservations). These results suggest that these cells are
crucial for setting the speed of the locomotor rhythm.
Indeed, adult En1 mutant mice that have defects in V1
connectivity are unable to perform fast stepping movements21. The ability to acutely inactivate neurons is especially important when the loss of a gene or a geneticallydefined cell type causes peri­natal death or major developmental defects that preclude behavioural analyses, as isoften the case for developmentally regulated genes. Suchapproaches will also facilitate functional analyses aimed
at dissecting more complex motor behaviours in awake
behaving animals40,100.
It is still not clear how the V1 neurons regulate thespeed of the locomotor step cycle. When cats are induced
to walk fictively, Renshaw cells and Ia inhibitory interneurons show an overlap in activity with the motor neurons
that they innervate, becoming active at the trans­ition
phase101. Although the inhibition provided by thesecells may contribute to burst termination during fictive
walking, excitatory neurons that provide rhythmic drivefrom the CPG are likely to be the predominant targetof V1 inputs. Intriguingly, the Xenopus homologues of theV1 neurons, aINs, are known to be differentially recruitedduring slow and fast swimming behaviours, being more
active at faster swimming speeds than slower speeds79.
At slow swimming speeds slow afterhyper­polarization
bursts mediated by Ca2+-activated K+ currents might
be the primary mechanism for burst termination5.
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REVIEWS
At higher speeds, early phase inhibition to the CPG
from aINs would probably facilitate burst termination,
thereby increasing the frequency of swimming movements. However, the role aINs79 and their zebrafish CiA
counterparts80 have in regulating the speed of swimming
movements still needs to be examined.

Evolutionary implications
The seemingly evolutionarily conserved role of V1
neurons in regulating the speed of the locomotor CPG
a

E9 Dorsal

rhythm indicates that certain neuronal modules may
have been preserved between the swimming and walking CPG. This reflects the close phylogenetic relationship between spinal neuron cell types in swimming
vertebrates and their terrestrial counterparts, which isparticularly apparent in the embryonic spinal cord79,80,102–
104(BOX 3; FIG. 4). What remains to be determined is how
the swimming CPG network has been reconfigured toenable limb-driven locomotion. The transition appears
to have been gradual, with amphibians and reptiles

E11

Pax3/7

Lhx2
pd1
Foxd3
pd2
pd3 Isl1

dI1

Pax2

dI4

pd5 Lmx1b

dI5

pd6

WT1

dI6

pV0 Evx1/2

V0

pd4

Pax6

Nkx6.1

dI2
dI3

En1

V1

pV2 Chx10/Gata3

V2

pV1

pMN
Hb9
pV3
Sim1

Sensory

Sonic hedgehog
BMP4,6,7, GDF-7
Motor

Nkx6.1
Pax6

MN
V3

Pax3/7

Ventralb
Embryonic

Adult cells

CINs,
GABA/Glycine

Disynaptic crossed
inhibitory INs?

dI6

WT1

V0

Evx1/2 V0V and V0D CINs,
2–4 segments, GABA/Glycine (V0D),
Glutamate (V0V)

Disynaptic crossed
commissural INs?

V1

En1

Ipsilateral rostral INs,
1–3 segments, GABA/Glycine

Renshaw cells,
Ia inhibitory INs

V2

Chx10
Gata3

Ipsilateral caudal INs, > 2 segments
V2a (Glutamate), V2b (GABA/Glycine)Unknown

MN

Hb9
Isl1

Peripheral projections,
Acetylcholine

Somatic/visceralmotor neurons

V3

Sim1

V3V and V3D
mixed commissural/ipsilateral> 2 segments (Glutamate)

Unknown

Adult
II
III
IV
V
VI
VII

I
DRG

IX
MN

Ia

X
Ia

VIII
V0 CIN RC

MN

MuscleFigure 3 | Early development of the spinal cord. a | Schematic cross-sections through the developingmouse
spinal cord
Nature Reviews| Neuroscienceshowing the patterning and specification of early spinal cord progenitors and their neuronal progeny. At embryonic day 9(E9), a gradient of Sonic hedgehog (red) (ventrally) and bone morphogenetic proteins (BMPs) and growth differentiation
factor 7 (GDF-7) (yellow) (dorsally) provide instructive positional signals to dividing progenitors in the ventricular zone. This
leads to the restricted activation of patterning factors in discrete dorsoventral domains, including Nkx6.1 (ventral), Pax6
(intermediate), and Pax3 and Pax7 (dorsal). At E11, eleven early classes of postmitotic neuron are present in the embryonicspinal cord. dI1-dI5 neurons that are derived from dorsal progenitors (grey) primarily contribute to sensory spinal pathways,
whereas dI6, MN and V0–V3 neurons arising from intermediate/ventral progenitors (yellow) are involved in the locomotor
circuitry. Some of the postmitotic transcription factors that serve to identify each of the eleven early generic populations
are indicated. b | Six classes of embryonic neurons are thought to give rise to the core elements of the spinal locomotor
circuitry. The neurotransmitter phenotypes and axonal projections of these embryonic neurons are indicated along withsome of the known adult cells types that are derived from each population. The image on the right is a schematic of theadult spinal cord showing the position and projections of somatic motor neurons (MN) (yellow), V1-derived Renshaw cells(RC) and Ia inhibitory interneurons (Ia) (green) and V0 commissural interneurons (CINs) (blue). The laminae of the spinal cord
are indicated by Roman numerals. DRG, dorsal root ganglion; GABA, γ-aminobutyric acid.
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REVIEWS
exhibiting standing wave flexion movements of the axial
body that are closely coupled to limb movements. It is
still unclear whether the emergence of limb movements
resulted from the reconfiguration of the swimming CPG
at limb levels or the addition of a specialized modulethat controls limb flexor–extensor muscle groups. Whatis clear is that both forms of locomotion can be present
in an animal at the same time, as in amphibia there is a
gradual shift in the mode of locomotion during larval toadult metamorphosis105.
The pectoral fins of teleosts possess primitive muscles that are homologous to dorsal (extensor) and ventral (flexor) limb muscle groups in walking vertebrates,
and the progenitors for these muscles express the Lbx1
transcription factor 106, which is selectively expressed in
the migrating appendicular muscle precursors of terrestrial vertebrates107,108. These observations, together withstudies in zebrafish showing walking-like movementsof the pectoral fins during slow swimming 109, suggestthat the neural substrates that are necessary for limbdriven locomotion are already present in bony fish.
Modelling studies are also supportive of this proposition110. For this reason, a detailed knowledge of the
motor circuitry in swimming vertebrates should provide important insights into how the walking CPG in
terrestrial vertebrates is organized.

New tools, challenges and vistas
Until now, genetic analyses of the circuits in the spinal
cord controlling locomotion have primarily used genetic
and fluorescent tracers to label and trace particular neuronal cell types32–36,58,59,80,95–97,103 (FIG. 5). In a few instances particular populations of neurons have been silenced either
by expressing channels that attenuate neuronal activity
or by blocking synaptic transmission35,36,40 (FIG. 5). Thesestudies have revealed an underlying modular organization of the locomotor CPG, however, the relatively simple
outputs measured in this way fail to reflect the richness
and complexity of motor behaviours that are elicited during vertebrate locomotion. Nonetheless, an increasingly
sophisticated arsenal of molecular and genetic tools thatcan be integrated with physiological and computational
analyses of motor networks is becoming available. Amongthese are a variety of channels that can be used to either
excite cells or to suppress neuronal activity and synaptic transmission36,40,99,100,111–115. Moreover, the spatial andtemporal expression of these channels in vertebrate systems can be further refined with the use of intersectional
approaches and tetracycline inducible promoters37–38,116.
The developmental genes that have been analysed todate are typically expressed in generic classes of neurons or in a spectrum of neuronal cell types. Future success will depend on identifying unique combinations

Zebrafish spinal cord
DoLA

CoPA
CiA

CoSA
VeLD

CoLo
CoLA

UCoD
MCoD

CoBL
CiD

VeMe

KA

VeSe

Embryonic mouse spinal cord
dl1

d15

d14

dl2

V0

dl3

dl6

dl3
V2b

V2a

V1
V3

Figure 4 | Identified spinal interneurons in the embryonic mouse and zebrafish spinal cord. Similar neuronal cell
Naturezebrafish
Reviews | Neuroscience
types are present in the embryonic spinal cords of aquatic and terrestrial vertebrates. The putativehomologuesof V0, V1, V2 and V3 locomotor interneurons are indicated by the same colour. These include V0 and CoSA neurons (lightblue), V1 and CiA neurons (dark green), V2a and CiD neurons (orange), V2b and VeLD neurons (turquoise), and V3, UCoD
and VeMe neurons (red).
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REVIEWSof genes that mark specific cell types. This is alreadypossible for Renshaw cells, as they are the only neurons in the cord that express both En1 and calbindin
D28K58. Hb9+/VGlut2+ neurons are also amenable togenetic manipulation (Refs 95,96) . However, there is
still a paucity of molecular markers that can be used tofunctionally subdivide these generic classes of spinal
interneurons. Genetic screens to identify genes thatare expressed in subsets of spinal interneurons are

beginning to address this deficit 117. The hope is thatthese efforts will identify new populations of loco­motor
interneurons and generate a coherent classification
system for the many functional interneuron subtypes
within the spinal cord.
New technologies are emerging for manipulatingwell-defined populations of neurons, and these should
lead to a better understanding of how the spinal locomotor system is organized and functions. Experimental

a Cre-dependent lineage marking
En1 promoter

En1 gene

ROSA26 promoter

Cre

V1 subtypes
En1Cre:ROSA26GFP

En1Cre:ROSA26IacZ
TTC-EGFP

GFP

MafB

Iacz or GFP

IoxP

Exon 2b V1 connectivity
IacZ

Stop

X

Exon 1IoxP

Renshaw cells
En1Cre:ROSA26lacZ
FoxP2

IacZ

calbindin

calbindin

P15

gephyrin

P15

Ia INsV1 INs

MNs
RCs

c AL activation of GIRK channels
AL
AL

d Cre-dependent expression of AlstR in V1 neurons
En1 promoter

AL

En1 gene

K+
Cre

AL

Exon 1

AlstR
γ

β
GTP
GDP

Exon 2

CAG promoter

GIRK

α

lacZ-stop
loxP

AlstR-IRES-GFP
loxP

e AL-induced silencing of V1 neurons in the spinal cord
+10 nM AL
I-L2
I-L5
50 pA

5s
1s

Reviews| Neuroscience
Figure 5 | Cre-dependent manipulation of V1 neurons in the mouse. a | Example of fateNature
mappingand lineage
tracingusing the Cre-loxP system. Mice carrying an En1Cre allele, in which the first exon of the En1 gene was replaced withsequences encoding Cre, were crossed with ROSA26 reporter mice that express either green fluorescent protein (GFP) or
lacZ after removal of a loxP-flanked stop sequence. b | Intramuscular injections of the retrograde tracer TTC-EGFP in
En1Cre:ROSA26lacZ mice show that V1 interneurons (INs) are synaptically connected to motor neurons (MNs)58 (left panel).
V1 neuron subtypes differentially express MafB (Renshaw cells (RCs), red) and FoxP2 (Non-Ia/RC interneurons, blue)(middle left panel). RCs are V1-derived neurons characterised by the expression of calbindin (middle right panel). RCs
uniquely possess large gephyrin clusters on their soma and proximal dendrites (right panel). c | Schematic showing
allatostatin (AL)-dependent activation of inwardly rectifying G-protein-activated inwardly rectifying potassium (GIRK)
channels in mammalian neurons. d | Cre-dependent expression of the allatostatin receptor (AlstR) in V1 neurons in mice.
e | Selective expression of AlstR-GFP in postmitotic V1 neurons at embryonic day 11 results in a reduction in V1 neuron
excitability (left). This slows down the locomotor rhythm, which phenocopies the defect seen when V1 neurons are
ablated (right). I-L2, second left lumbar root; I-L5, fifth left lumbar root. Part e is reproduced, with permission, from REF. 36
 (2006) Macmillan Publishers Ltd. All rights reserved.

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REVIEWS
approaches to assess the temporal activity of neurons
within the locomotor CPG network during locomotion
are also needed to complement ongoing genetic andbehavioural analyses. Technologies that allow the visualization of, and recordings from, multiple neurons during
particular motor behaviours are now available118,119, andare currently being used to study swimming behaviours
in zebrafish embryos120–122. With respect to the walking
CPG, the neonate mouse spinal cord is perhaps ideal forsuch optogenetic analyses, owing to its small size, partial transparency and the ability to genetically identify
different neuronal populations using mouse molecular
genetic techniques.
Although the isolated spinal cord can provide
important insights into the motor circuitry, such preparations do not allow an assessment of the role thatparticular neurons have in postural control or complex motor behaviours. Many of the differentiated cell
types that are present in the mature spinal cord andhave specialized roles in sensorimotor reflexes cannot
be assessed in the neonate spinal cord as they are notfully differentiated and the sensory reflex pathways arestill immature. Consequently, developing novel genetic
approaches and behavioural tests that can be used forin vivo experimentation in adults will be necessary
and can be guided by previous efforts in the cat. Other
drawbacks with mice include difficulties in activating
specific sensory reflexes and stimulating descending pathways, as well as recording from neurons in
the intact cord. These issues are not insurmountable and efforts are underway to record from spinal
inter­neurons in adult mice.
Another important issue is whether genetic markers
represent a valid approach for identifying different

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Acknowledgements

Studies from the author’s laboratory were supported by
grants from the US National Institutes of Health (NS31978,
NS37075 and NS31249), the Human Frontier Science
Program and the Christopher Reeve Paralysis Foundation.
Many thanks to D. McLean and J. Fetcho for the zebrafish
schematic in figure 4. I apologize to those whose work I was
unable to fully cite because of space constraints.

Competing interests statement

The author declares no competing financial interests.

DATABASES
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=gene
En1 | glrb2 | Pax6
OMIM: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=OMIM
UniProtKB: http://ca.expasy.org/sprot
calbindin D28K | Chx10 | Dbx1 | Epha4 | ephrin B3 | GlyT1 |Nck adaptor protein | Shh

FURTHER INFORMATION
Martyn Goulding’s homepage: http://www.salk.edu/
faculty/goulding.html
All links are active in the online pdf

www.nature.com/reviews/neuroo n l i n e o n ly
Author Biography
Martyn Goulding is a professor at the Salk Institute for BiologicalStudies in La Jolla, California, USA. After obtaining his Ph.D. from the
University of Auckland, New Zealand, he moved to the Max Planck
Institute for Biophysical Chemistry in Goettingen, Germany. There heworked in the laboratory of Peter Gruss, studying the role of the Paxtranscription factors in development. His current interests are focusedon the development of sensorimotor circuits in the spinal cord andon ascertaining how these circuits are organized and function at thecellular level.
TOC blurb

controlling vertebrate locomotion:000		 Circuits
moving in a new direction
Martyn Goulding
Intrinsic spinal cord networks generate the rhythmic
patterns of motor activity that underlie locomotion.
Goulding shows how genetic analyses, coupledwith classical systems neuroscience approaches,are providing new information about the cellularcomponents and functional organization of thesecircuits.
• The central pattern generator (CPG) networks that generate relatively simple motor outputs are ideal experimental models for circuit analysis.
• Locomotor CPGs in the ventral spinal cord function autonomously
to generate repetitive patterns of oscillatory motor activity.
• Recent progress has been made in identifying the neuronal components that make up the locomotor circuitry, with functional studies
indicating that the locomotor CPG has a modular structure.
• The development and assembly of the locomotor CPG is regulated by a genetic programme that operates in the embryonic spinal
cord.
• The merging of genetic analyses with systems approaches, coupled with new tools for imaging and regulating neuronal excitability, provides the means for a comprehensive analysis of thesecircuits.
• The emerging phylogenetic relationship between neurons in thevertebrate spinal cord is providing key insights into the structureand function of the spinal motor circuitry.