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ANRV278-NE29-10 ARI 8 May 2006 15:58
Locomotor Circuits in theMammalian Spinal CordOle KiehnMammalian Locomotor Laboratory, Department of Neuroscience, KarolinskaInstitutet, Stockholm S17177, Sweden; email: [email protected]
Annu. Rev. Neurosci.2006. 29:279–306
The Annual Review ofNeuroscience is online atneuro.annualreviews.org
doi: 10.1146/annurev.neuro.29.051605.112910
Copyright c© 2006 byAnnual Reviews. All rightsreserved
0147-006X/06/0721-0279$20.00
Key Words
motor control, interneurons, transcription factors, moleculargenetic, transgenic mice
AbstractIntrinsic spinal networks, known as central pattern generators(CPGs), control the timing and pattern of the muscle activity under-lying locomotion in mammals. This review discusses new advances inunderstanding the mammalian CPGs with a focus on experimentsthat address the overall network structure as well as the identifi-cation of CPG neurons. I address the identification of excitatoryCPG neurons and their role in rhythm generation, the organizationof flexor-extensor networks, and the diverse role of commissuralinterneurons in coordinating left-right movements. Molecular andgenetic approaches that have the potential to elucidate the functionof populations of CPG interneurons are also discussed.
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Contents
INTRODUCTION. . . . . . . . . . . . . . . . . 280LOCALIZATION OF THE
LOCOMOTOR CENTRALPATTERN GENERATOR. . . . . . . 281Longitudinal Distribution . . . . . . . . 281Transverse Distribution . . . . . . . . . . . 283Consequences for Central Pattern
Generator Layout . . . . . . . . . . . . . 283KEY FEATURES OF THE
WALKING CENTRALPATTERN GENERATOR. . . . . . . 283
EXCITATORYRHYTHM-GENERATINGCIRCUITS . . . . . . . . . . . . . . . . . . . . . . 283Four Classes of Excitatory
Interneurons Have BeenIdentified . . . . . . . . . . . . . . . . . . . . . 284
Role in Rhythm Generation. . . . . . . 285CIRCUITS INVOLVED IN
FLEXOR-EXTENSORCOORDINATION . . . . . . . . . . . . . . 289
CIRCUITS INVOLVED INLEFT-RIGHTCOORDINATION . . . . . . . . . . . . . . 291Anatomical Organization . . . . . . . . . 292Activity in Descending
Commissural Interneurons areInvolved in Binding SynergiesAcross the Cord . . . . . . . . . . . . . . . 293
Role of Ascending CommissuralInterneurons . . . . . . . . . . . . . . . . . . 294
Functional Organization ofIntrasegmental CommissuralInterneurons . . . . . . . . . . . . . . . . . . 294
Commissural Interneurons andRhythm Generation . . . . . . . . . . . 297
GENETIC NETWORKCRACKING. . . . . . . . . . . . . . . . . . . . . 297
SUMMARY ANDPERSPECTIVES . . . . . . . . . . . . . . . . 298
INTRODUCTION
Locomotor behaviors (such as flying, swim-ming, or walking) are fundamental motoracts that give animals and humans the abil-ity to move. Such motor acts involve theactivation of many muscles. Localized neu-ronal networks called central pattern genera-tors (CPGs) generate much of the timing andpattern of these complex, rhythmic, coordi-nated muscle activities. CPGs controlling lo-comotion are located in the spinal cord andare found in all vertebrates, including humans.When locomotion starts, activity in the CPGis turned on and maintained by inputs fromdescending locomotor commands originatingfrom neurons in the brainstem and midbrain.However, it is the neurons in the locomo-tor CPG that generate the rhythm and pat-tern of muscle contraction. Research over thelast 20–25 years in two nonmammalian verte-brate species, the lamprey and the Xenopus tad-pole, has provided a detailed network struc-ture of CPGs controlling swimming as well asmany of the details of the cellular and synap-tic mechanisms of swimming CPG functionand its modulation (Grillner 2003, McLeanet al. 2000, Roberts et al. 1998). Comparedwith this extensive knowledge, less is knownabout locomotor CPGs in mammals (Claracet al. 2004, Hultborn et al. 1998, Kiehn &Butt 2003, McCrea 1998), despite the factthat the walking CPG in mammals was firststudied almost 100 years ago (Brown 1911).New knowledge on the mammalian locomo-tor CPGs is, however, advancing rapidly.
Here I discuss some of these advances witha focus on experiments that address the over-all structure of the spinal locomotor networksas well as the identification of CPG neurons.Such knowledge is the foundation for under-standing the function of the mammalian lo-comotor CPGs, including how the brain canactivate the network and how cellular andsynaptic properties as well as neuromodula-tion contribute to the rhythmicity. Because ofthe large number of cells present in the mam-malian spinal cord, this knowledge cannot
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be simply obtained with the classical electro-physiological and anatomical techniques thathave been successfully used in smaller mo-tor systems to crack the network structure.This review therefore also includes discus-sions of new molecular and genetic strategiesthat, when combined with a classical approachto network analysis, hold promise to give un-precedented insight into the network struc-ture. In this aspect locomotor CPGs are idealmodel systems for studies of complex neu-ronal networks because they produce mea-surable outputs that directly relate networkactivity with actual behavior. Moreover, thebasic mechanism for CPG function mightvery well be conserved in cortical networksthat control more complex behaviors (Yusteet al. 2005). Understanding the locomotorCPG in mammals is also an important step inimproving clinical neuro-rehabilitation of pa-tients with spinal cord injury (Edgerton et al.2004, Rossignol et al. 1998). Without under-standing the neuronal circuits in the spinalcord that produce motor behaviors, it is dif-ficult to design better therapies to improvethem after spinal lesions.
LOCALIZATION OF THELOCOMOTOR CENTRALPATTERN GENERATOR
Early studies showed that the lumbar spinalcord contains sufficient neuronal elements toproduce the precise timing and activation ofthe large number of hindlimb muscles ac-tive during locomotion (see Grillner 1981).Similarly, forelimb locomotion is controlledby locomotor networks located in the cervi-cal spinal cord. What these initial studies didnot answer, however, is the following: Whatis the extent of the CPG circuits in the lum-bar or cervical spinal cord in the longitudinaland transverse planes? This apparently simplequestion has fostered a large number of stud-ies over the years and led to conflicting resultsregarding the distribution of the spinal loco-motor network. Most studies have focused onthose networks controlling hindlimb locomo-
CPG: centralpattern generator
Rhythmogeniccapacity: alludes tothe capability ofCPG elements togenerate a rhythm
Drug-inducedrhythmic activity:the isolated spinalcord of newbornrodents can producea rhythmic motoroutput when exposedto rhythmogenictransmitter agonistssuch as 5-HT,NMDA,noradrenalin, anddopamine, alone orin combinations
tion, and the discussion here is restricted tothese studies.
Longitudinal Distribution
Grillner & Zangger (1979) first addressed therostrocaudal extent of the rhythmogenic ca-pacity of the hindlimb locomotor CPG net-work in mammals by using transverse section-ing of the cord in the cat. Alternating rhythmicactivity could be evoked in ankle flexors andextensors when the caudal lumbar cord, theL6–S1 segments, was isolated from the restof the cord, suggesting the rhythmogenic ca-pacity in the CPG controlling hindlimb loco-motion is distributed throughout the lumbarenlargement (L3–S1 in cats). Results from anumber of different laboratories using the iso-lated spinal cord preparation from newbornrodents (rats or mice) and studying sponta-neous or drug-induced rhythmic activity be-fore and after transverse trans-sectioning atdifferent spinal levels concur with this idea(Bonnot & Morin 1998, Bonnot et al. 2002,Bracci et al. 1996a, Christie & Whelan 2005,Cowley & Schmidt 1997, Gabbay et al. 2002,Kjaerulff & Kiehn 1996, Kremer & Lev-Tov1997, Kudo & Yamada 1987). Similar re-sults have also been obtained for sponta-neous rhythmic activity in the chick (Ho &O’Donovan 1993) and for rhythmic scratch-ing in the turtle (Mortin & Stein 1989) andcat (Deliagina et al. 1983).
What is clear from all of these experi-ments, however, is that there is a differencebetween rostral and caudal segments in theirrhythmogenic capacity. Rostral lumbar seg-ments (L1–L3 in rodents, L3–L5 in cats, andD7–D10 in turtles) have a greater capacityto generate rhythmic motor output in isola-tion than caudal segments (L4–L6, L6–S1,and S1–S2, respectively). Together these stud-ies suggest that the rhythmogenic capacity ofthe mammalian hindlimb locomotor CPG isdistributed along the lumbar cord but witha rostrocaudal excitability gradient. A greaterproportion of intraspinal inputs to rostral seg-ments than to caudal segments may be one
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5-HT:5-hydroxytryptamine
NMDA:N-methyl-d-asparticacid
cause of this gradient (Berkowitz 2004). An-other possibility is differential modulation ofrhythmogenic networks in the rostral andcaudal cord by neuromodulatory substances.A recent study has shown that 5-hydroxy-tryptamine (5-HT) and dopamine, when usedtogether in concentrations that produce sta-ble locomotor activity, set up a rostrocaudalgradient of excitability in the mouse spinalcord, possibly owing to differential distribu-tion of receptors or receptive neurons alongthe cord (Christie & Whelan 2005). Thesefindings suggest a rhythmogenic network witha rostrocaudal gradient is a requirement forproducing a stable locomotor output.
A different image of the rhythmogenic ca-pacity in the hindlimb enlargements in mam-mals emerged from studies in the neonatal ratusing a partitioning setup, where the upperand lower lumbar cord could be selectivelyexposed to rhythmogenic drugs (Bertrand &Cazalets 2002, Cazalets et al. 1995). Thus,when the upper lumbar enlargement (L1–L2)was exposed to a combination of 5-HT andN-methyl-d-aspartic acid (NMDA), rhyth-mic locomotor-like activity could be recordedin upper as well as in lower lumbar segments(although weaker in the lower), whereas whenthe same combination and concentration ofdrugs were both applied to the lower lumbarcord (L3–L6), only tonic activity was inducedin lower lumbar segments. These observa-tions led the authors to suggest that spinalinterneurons directly involved in producingrhythmic activity are restricted to the T13and L2, whereas the lower segments, wheremost of the motor neurons innervating thehindlimbs are located, have no rhythmogeniccapacity. A similar conclusion was reachedfrom experiments in intact adult rats. Whenthe gray matter in L1–L2 was destroyed bykainate injection, the overground locomo-tor capability was greatly impaired whereasa kainate injection more caudally had muchless of an effect on the locomotor capabilityof the intact rat (Magnuson et al. 2005). Thisnotion was qualified further in experiments inchronic spinal cats sectioned at Th13 (Lan-
glet et al. 2005). Such cats can be made towalk on a treadmill after extensive trainingand simultaneous stimulation with clonidine,a noradrenergic agonist, to improve the spinallocomotor capacity (Langlet et al. 2005). Inthese experiments it was shown that the abilityto walk under these conditions was lost after asecond transverse sectioning at L4, indicatingthe midlumbar region (L3–L4) rostral to thehindlimb motor neurons is essential for theexpression of locomotion.
How can these discrepancies be explained?As previously discussed (Kiehn & Kjaerulff1998) it is likely the physical extent of therhythmogenic capacity is dependent on thetype and/or concentration of the transmit-ter agonists used to induce rhythmic activity.For example, in the rodent 5-HT alone canproduce alternating rhythmic activity in thehindlimb when applied to the lower thoracic–upper lumbar spinal cord but not to the lowerlumbar cord (Cowley & Schmidt 1997). Incontrast, low concentrations of 5-HT [lowerthan those used in the partitioning experi-ments by Cazalets et al. (1995)] in combina-tion with NMDA, acetylcholine in combina-tion with an acetylcholine esterase inhibitor,or noradrenalin alone are capable of induc-ing rhythmic activity in isolated parts of boththe rostral and caudal lumbar cord (Cowley &Schmidt 1997, Gabbay et al. 2002, Kjaerulff& Kiehn 1996). The likely explanation forthese differences in rhythmogenic capacity inthe newborn rodent is variation in the post-synaptic receptor distributions on spinal neu-rons (Liu & Jordan 2005, Schmidt & Jordan2000), which would provide sufficient expla-nation for the reported differences. With re-gard to the experiments in adult rats (Mag-nuson et al. 2005) or cat (Langlet et al. 2005),the loss of hindlimb locomotion after destroy-ing the rostral lumbar cord or disconnect-ing it from caudal cord does not necessar-ily suggest that rostral segments contain allthe rhythmogenic neurons needed to gen-erate locomotion. An alternative explanationis that these segments contain rhythmogenicCPG interneurons that directly control hip
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movements. Without hip flexion the leg can-not be cleared off the ground, and overgroundwalking will be severely impaired.
Transverse Distribution
Although there has been much discussionabout the rostrocaudal extent of the rhyth-mogenic capacity, there is little disagreementabout the distribution in the transverse plane.Activity-labeling studies (Cina & Hochman2000, Dai et al. 2005, Kjaerulff et al. 1994)and electrophysiological evidence (see Tresch& Kiehn 1999) show that locomotor-relatedneurons are concentrated in a ventral location(laminae VII, VIII, and X), suggesting all thecritical elements of the locomotor circuit inmammals are located there. This notion hasbeen directly confirmed in microlesion stud-ies in the rodent (Bracci et al. 1996a, Kjaerulff& Kiehn 1996). Therefore, all studies of theneuronal elements in the mammalian CPGconcentrate on the ventral spinal cord.
Consequences for Central PatternGenerator Layout
Taken together, the available evidence onthe rhythmogenic capacity in the mammalianhindlimb CPG may be synthesized into oneunified model: The rhythmogenic capacity islocated ventrally in the cord and is distributedover the lumbar spinal cord and, at least in therodent, into the lower thoracic spinal cord,with a greater capacity to generate a rhythmin rostral than in caudal segments. This ros-trally biased organization is also found inother limbed vertebrates, such as the chick andturtle. The organizational principle suggeststhat instead of having one rhythm-generatingcore localized in the upper lumbar cord, themammalian locomotor CPG is composed ofmultiple distributed rhythm-generating corenetworks /modules as originally proposed byGrillner (1981). This segmental organiza-tion would then be similar to what has beenfound in fish (Grillner 2003) and what hasbeen suggested for rhythmic scratching in
turtles (Stein 2005). The rhythm-generatingcore networks appear to be recruited into afunctional unit forming one rhythmic net-work when locomotion is initiated, whichwould explain why evidence for independentburst generation is not seen under most ex-perimental conditions (Lafreniere-Roula &McCrea 2005), unless the cord is trans-sectioned. That the rhythmogenic capacityis highest in the rostral cord where hip mo-tor neurons are located indicates the rhyth-mogenic network controlling hip movementacts as a leading oscillator, entraining morecaudal and less excitable oscillators, for ex-ample, those controlling the knee and ankle(see Stein 2005 for a discussion). Such cou-pled networks have been previously describedfor the rhythm-generating networks control-ling the circadian rhythm (Albrecht & Eichele2003) and respiration (Mellen et al. 2003).
KEY FEATURES OF THEWALKING CENTRAL PATTERNGENERATOR
The key features of walking are (a) therhythm, (b) the ipsilateral coordination of flex-ors and extensors across the same or differentjoints in a limb, and (c) left/right coordination.These functions are all integrated in the fullyfunctioning CPG. It might, however, be use-ful conceptually to consider these functionsas generated by separate network structuresor neuronal populations. This tripartite struc-ture is considered in separate sections below:(a) excitatory rhythm generating circuits, (b)circuits involved in flexor-extensor coordina-tion, and (c) left-right coordination circuits.
EXCITATORYRHYTHM-GENERATINGCIRCUITS
Ipsilaterally projecting last-order gluta-matergic excitatory interneurons are mostlikely the source of rhythm generation inthe tadpole and lamprey swimming CPGs(Grillner 2003). These interconnected,
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Brainstem andmidbrainlocomotor regions:regions in thebrainstem andmidbrain that whenstimulatedelectrically activatethe spinal locomotorCPGs
EphA4: ephrinreceptor A4
EphrinB3: ephrinligand B3
excitatory neurons are distributed segmen-tally along the cord and seem to function asburst-generating units that provide rhythmic,excitatory, glutamatergic synaptic drive tomotor neurons and other ipsilateral in-hibitory and left-right coordinating CPGneurons in each segment. Experimentsblocking inhibitory synaptic mechanisms inthe lamprey hemicord show that crossedinhibitory activity and ipsilateral inhibitoryinterneurons are not needed for rhythmgeneration (Cangiano & Grillner 2003,2005). Similar results have been obtained inthe rodent (Bonnot et al. 2002, Bracci et al.1996b, Kjaerulff & Kiehn 1997, Kremer &Lev-Tov 1997) and cat (Kato 1987, Nogaet al. 1987). This suggests excitatory networksare responsible for rhythm generation inthe mammalian spinal cord and exclude ahalf-center organization where interneuronsrelated to flexor activity and interneuronsrelated to extensor activity are lumped intotwo modules and the reciprocal inhibitionbetween these half-center modules is respon-sible for the rhythm (see Stein & Smith 1997for a discussion of other reasons to discardthe half-center model). Until now knowledgeabout the identity of excitatory interneuronsin the mammalian spinal cord and theirinvolvement in generating locomotion is,however, sparse.
Four Classes of ExcitatoryInterneurons Have Been Identified
Interneurons located in the lower lumbosacralregion in the intermediate area of the spinalcord that project to extensor motor neuronsin the same segment have been found to berhythmically active during locomotion in thecat (Angel et al. 2005). Based on their firingpattern and activation from extensor group Iafferents (muscle spindles and tendon organs),they were proposed to be last-order excita-tory interneurons and involved in supportingthe extensor phase during locomotion. Di-rect evidence that these neurons are excita-tory is, however, missing. Another group of
potential excitatory CPG interneurons in thecat belongs to a population of neurons lo-cated in the intermediate zone in the mid-lumbar region (L3–L4). Using nonlocomot-ing preparations, Jankowska and colleaguesoriginally characterized these neurons thatreceive strong inputs from group II muscleafferents, project to motor neurons locatedin the lower (L7) lumbar cord, and includelast-order inhibitory and excitatory neurons(Cavallari et al. 1987; Edgley & Jankowska1987a, 1987b). Shefchyk et al. (1990) showedthat two-thirds of the group II cells are rhyth-mically active in the flexor phase during loco-motion and are activated from the brainstemlocomotor regions. Unfortunately Shefchyket al. (1990) did not determine whether thecells they recorded from were excitatory or in-hibitory. It is therefore difficult to determineto what extent the last-order excitatory mid-lumbar group II interneurons can be classifiedas locomotor-related interneurons.
Two series of studies using geneticallymodified mice have identified two other pop-ulations of putative mammalian excitatoryCPG interneurons. The first series of stud-ies used mice with targeted deletions inthe genes for the axon guidance moleculesephrin receptor A4 (EphA4) and ephrin lig-and B3 (ephrinB3) to identify a group of ex-citatory spinal interneurons rhythmically ac-tive during locomotion. ephA4 and ephrinB3knockouts display a characteristic hopping,rabbit-like gait (Coonan et al. 2001; Dottoriet al. 1998; Kullander et al. 2001a, 2001b;Yokoyama et al. 2001), which is because of agenetic reconfiguration of the walking CPG(Kullander et al. 2003) (Figure 1a–c). EphA4-positive neurons, whose cell bodies are locatedin the spinal cord, aberrantly cross the mid-line in ephA4 and ephrinB3 knockouts (Kullan-der et al. 2003), apparently because ephrinB3forms a midline barrier that normally pre-vents EphA4-positive spinal neurons fromcrossing the midline. When the inhibitorydrive is chemically increased in mutant spinalcords, the synchronous hindlimb activity re-verts to a normal, alternating pattern. These
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observations led to the hypothesis that atleast some EphA4-positive spinal neurons areexcitatory CPG neurons, whose projectionsnormally remain ipsilateral but in mutantscross the midline and bind the activity ofthe two sides into synchrony. Recording fromEphA4-positive cells has now demonstratedthat the majority of interneurons expressingthe EphA4 receptor in the ventral region ofthe mouse lumbar spinal cord are rhythmi-cally active during locomotion (Butt et al.2005) (Figure 1f ). A subset of these neuronsprovides mono- or polysynaptic excitation ofmotor neurons in the same segment (Figure1e,h) and fires in an appropriate phase (Figure1g) as to be involved in generating rhythmic,excitatory drive to segmental, ipsilaterally lo-cated motor neuron pools.
The second series of experiments has takenadvantage of a transgenic mouse line wheregreen fluorescent protein is expressed un-der the control of the transcription factorHB9 (basic helix-loop-helix domain contain-ing, class B, 9) (Hinckley et al. 2005, Wilsonet al. 2005). It has been known for sometime that HB9 is expressed in motor neu-rons during embryonic life. The present stud-ies show that HB9 protein is also expressedin a small cluster of interneurons close tothe midline in the upper lumbar spinal cord(L1–L3) (Figure 2a). HB9 cells express thevesicular glutamate transporter, VGLUT2(Figure 2b), and they are rhythmically ac-tive during locomotion (Figure 2c). At themoment there is disagreement as to whetherthe HB9 interneurons make monosynaptic(Hinckley et al. 2005) or polysynaptic connec-tions (Wilson et al. 2005) with motor neurons.Despite these discrepancies and the fact thatit has not been shown directly that HB9 cellsprovide excitation of motor neurons, the over-all picture of the HB9-positive interneuronsstrongly suggests they are involved in provid-ing excitation to motor neurons during loco-motion (Figure 2d ).
As the HB9 cluster is located more me-dially in the rodent spinal cord than rhyth-mically active, excitatory EphA4 positive cells
Mono- andpolysynaptic: oneand two or moresynapses
HB9: basichelix-loop-helixdomain containing,class B, 9
are, it is likely these excitatory neurons consti-tute two distinct neuronal populations. More-over, the two rodent populations share fewcommon characteristics in terms of laminarlocation and/or projections with the two neu-ronal populations described in the cat. Thiswould imply, unlike the case in lamprey andtadpole, that more than one population of ex-citatory interneurons is involved in making upthe mammalian locomotor CPG.
Role in Rhythm Generation
A pertinent question arising from these stud-ies is if any of these interneuron popu-lations are directly involved in generatingthe rhythm. The group I–activated extensor-related interneurons lose their rhythmicityand become tonically active when flexor mo-tor neurons momentarily drop out of the lo-comotor rhythm (owing to a decreased orabsent drive from the flexor CPG). Thisbehavior precludes that these interneuronsare the prime drivers of the extensor mo-tor neuron activity during locomotion. Theirrole seems to be to gate excitatory activ-ity from movement-activated extensor mus-cle receptors onto extensor motor neuronsduring the stance phase and thereby con-tribute to the amplitude of the motor out-put. Edgley & Jankowska (1987b) proposedthe midlumbar neurons to be involved inextensor-flexor transition during locomotion.Transition from stance (extension) to swing(flexion) happens when the hip is extended toan 80–90◦ angle (Grillner & Rossignol 1978)and is influenced by sensory inputs from hipmuscle and joint afferents. The midlumbar in-terneurons receive multisensory inputs fromthe muscles stretched at the end of the stancephase, and these interneurons may contributeto phase transition by way of the inhibitionof extensors and/or the excitation of flexors.As reported by Shefchyk et al. (1990), thefiring in the flexor phase for the midlumbarinterneurons is compatible with this hypoth-esis, suggesting at least some of the midlum-bar interneurons provide rhythmic excitation
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of motor neurons during locomotion. But arethey involved in generating the rhythm itself?It is impossible to answer this question di-rectly for the midlumbar as well as for theHB9- and EphA4-positive populations at themoment because a proof of causality is lacking(see Genetic Network Cracking, below). In-formation about the overall layout of the loco-motor CPG can, however, provide importantadditional clues as to whether these or anynew populations of excitatory interneuronsthat might be described are directly involved
in rhythm generation. For example, the dis-tribution of rhythmogenic capacity along thecord precludes groups of locally projectingneurons restricted to one part of the cord suchas the HB9 cluster as the sole source of rhythmgeneration in the hindlimb locomotor CPG.
To this end it is of interest to consider therecent proposal that the mammalian locomo-tor CPG has two layers: a rhythm-generatinglayer separated from a pattern-generatinglayer (coordinating flexor-extensor and left-right side activity) (Burke et al. 2001,
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EphA4-positiveinterneuron
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Kriellaars et al. 1994, Lafreniere-Roula &McCrea 2005). In this two-layer model theneurons in the pattern-generating layer areprojecting monosynaptically to motor neu-rons whereas the neurons in the rhythm-generating layer are two or several synapsesupstream from motor neurons and projectdirectly to pattern-generating neurons. Thislayout significantly differs from the one-layer layout found in swimming CPGs whererhythm and pattern are embedded in a one-layer CPG (Grillner 2003, Roberts et al.1998). Obviously, if the two-layer model iscorrect for the mammalian locomotor CPG,any rhythmically active excitatory interneu-rons with monosynaptic connections ontomotor neurons would be excluded from beinginvolved directly in rhythm generation andwould belong to the pattern-generating layer(see Wilson et al. 2005). Conversely, rhythmi-cally active excitatory neurons with polysyn-aptic connections to motor neurons would be
more likely candidates for being involved inrhythm generation in this model. The exper-imental evidence that has led to the proposalof the two-layer layout is threefold: (a) affer-ent perturbation can change motor pattern ac-tivity without influencing the rhythm (Burkeet al. 2001), (b) a change of amplitude in loco-motor output can happen independently of achange in locomotor speed (Kriellaars et al.1994), and (c) “deletions” of muscle activ-ity can be seen in individual muscles with-out resetting changes occurring in locomo-tor frequency (Lafreniere-Roula & McCrea2005). In all cases the argument is that thepattern can be changed independently of therhythm. The observed motor output changescan, however, also be interpreted in a one-layer model: (a) independent regulation ofmotor output amplitude can occur as a con-sequence of presynaptic modulation or ampli-tude modulation in the rhythmogenic popula-tion of neurons, and (b) deletions and changes
←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 1The axon-guidance receptor EphA4 functions as a molecular marker for excitatory central patterngenerator (CPG) neurons. (a–c) Mice with a targeted deletion in their EphA4 receptor or EphrinB3ligand systems have a specific motor phenotype: a rabbit-like gait with synchronous hindlimb movementsinstead of the normal left-right alternation. (a) Experimental set-up. Suction electrodes recorded motoractivity in four lumbar roots. (b and c) Transmitter-induced (NMDA/5-HT) locomotor activity in theisolated spinal cord from knockout mice reproduces the rabbit-like gait with segmental synchrony(rL2–lL2/rL5–lL5) and ipsilateral flexor-extensor alternation (e.g., rL2/rL5). (c) Normal locomotor-likepattern in control animals in panel b. (d) Experimental set-up used for experiments depicted in panels e–g.Suction electrodes recorded motor activity in three ventral roots: the contralateral lumbar segment 2(cL2), ipsilateral L5 (iL5)—both AC filtered—and ipsilateral L2 (iL2)—DC (direct current) filtered.Interneurons were in the ventral horn of the iL2 (gray). (e) Spike-triggered averaging of EphA4-positiveinterneurons revealed in some cases excitatory responses in ipsilateral L2 motor neurons (seen asdepolarizing DC averaged response in the thick trace below the spike). This response was blocked byeither incubation (20 min) in low Ca2+ Ringer’s (thin line) or the glutamatergic blockers CNQX(6-cyano-7-nitroquinoxaline-2,3-dione) plus AP5 [D(−)-2-amino-5-phosphonopentanoic acid].( f ) Rhythmically active EphA4-positive interneurons during drug-induced locomotion (NMDA plus5-HT). Instantaneous firing frequency over the locomotor cycle in lower panel. (g) Circular plot showingthe phase and rhythmicity of excitatory (black circles), inhibitory (white circles), and no signal (gray circles)interneuron classes that were found EphA4 positive. The gray shaded area represents the ipsilateral phaseof motor activity. Note that the excitatory EphA4 cells fire in phase with the ipsilateral motor neurons.(h) Schematic diagram of the EphA4 interneuron projections. The arrow crossing the midline indicatesthat in EphA4 or ephrinB3 mutant mice the excitatory EphA4 cells might, in addition to their ipsilateralprojections, have an axons crossing in the midline binding the segmental activity into synchrony. Data inpanels a–c adapted from Kullander et al. (2003) (with permission from AAAS, copyright 2003) and Kiehn& Butt (2003) (with permission from Elsevier, copyright 2003). Data in panels d–g adapted from Buttet al. (2005) (with permission from the National Academy of Sciences, copyright 2005).
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Figure 2Summary diagram of HB9 interneuron anatomy and physiology. (a) Fluorescent micrographs of rostrallumbar spinal cord section showing GFP-expressing motor neurons (ellipse), sympathetic preganglionicmotor neurons (circle), and the bilaterally located HB9 clusters (arrows). (b) HB9 cluster interneurons areglutamatergic as is revealed by in situ hybridization of VGLUT2 mRNA. Scale bar: 100 μm.(c) Rhythmic activity in HB9-GFP interneurons (locomotion induced by combination of 5-HT, NMDA,and dopamine. (d) Schematic diagram of the HB9 cluster interneurons in the mammalian central patterngenerator (CPG). (e) When all synaptic inputs are blocked with TTX (tetrodotoxin), Hb9 clusterinterneurons could display large membrane oscillations when exposed to locomotor drugs[NMDA-5-HT-DA (dopamine)]. The frequency of these oscillations was voltage dependent. Panels a, b,and e adapted with permission from Wilson et al. (2005) (with permission from the Society forNeuroscience, copyright 2005). Panel c adapted from Hinckley et al. (2005) (with permission from theAmerican Society for Physiology, copyright 2005).
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in motor pattern owing to afferent perturba-tion can be explained in CPG models withmultiple burst generators (Stein 2005) whererhythm-generating neurons momentarily fallsubthreshold for spiking but maintain theiroscillatory activity (deletions) or are recruitedin or out of the network by afferent input.It seems premature, therefore, to ascribe arole for excitatory neurons in rhythmogenesisbased on mono- or polysynaptic connectivityto motor neurons.
There are also connectivity patterns andcellular properties that might provide indi-rect clues as to whether a population of ex-citatory cells is involved in rhythm gener-ation. A general concept that has emergedfrom studies of the excitatory CPG neurons inthe tadpole (Roberts et al. 1998), the lamprey(Grillner 2003), and the mammalian respira-tory network (Smith et al. 2000) is that theexcitatory interneurons are interconnected.Such an excitatory network can generate pro-longed, persistent output when appropriatelyactivated from an external source. For theHB9 cells anatomical evidence exists for suchinterconnectivity (Figure 2d ), whereas noneof the other cell populations described in thissection have been investigated in this way.Because the locomotor rhythm can be gen-erated in the absence of inhibition, excita-tory CPG neurons may have some intrinsicpacemaker-like properties (Pena et al. 2004,Smith et al. 2000) or some other set of voltage-dependent membrane conductances that sup-port rhythmic firing. Early studies in the ro-dent have shown that a minor percentageof unidentified neurons located in the ven-tromedial region close to the central canalcan possess conditional pacemaker proper-ties induced by certain rhythmogenic sub-stances such as NMDA, 5-HT, and mus-carine (Hochman et al. 1994, Kiehn et al.1996). Similar to these findings a subpopula-tion of the HB9-positive interneurons showspacemaker properties or rhythmogenic ionicconductances after the application of rhyth-mogenic transmitter agonists such as NMDA,
Pacemakerproperties: thecapability of neuronsto generatemembrane potentialoscillations in theabsence of extrinsicinputs
Commissuralinterneurons(CINs): neuronsthat have axonscrossing the midline
Ia interneurons(Ia-INs): glyciner-gic/GABAergicipsilaterallyprojecting inhibitoryinterneurons
Renshaw cells(RCs): glyciner-gic/GABAergic,ipsilaterallyprojecting inhibitoryinterneurons
5-HT, and dopamine (Figure 2e). Whetherthe EphA4 neurons have such properties isunknown.
Another prediction from the lamprey andtadpole CPG network is that that the ex-citatory CPG responsible for the rhythmconnects to all other CPG interneurons. Itwill therefore be as important to track con-nections to other spinal interneurons as tomotor neurons to understand the function ofthe rhythm-generating network in the mam-malian locomotor CPG.
CIRCUITS INVOLVED INFLEXOR-EXTENSORCOORDINATION
Appropriate alternation between flexor andextensor motor neurons on the same side re-quires inhibitory networks. Thus flexor andextensor motor neurons receive rhythmicglycinergic inhibition alternating with rhyth-mic glutamatergic excitation (see Kiehn et al.1997, and references therein). When glyciner-gic inhibition is blocked, flexors and extensorsare activated in synchrony (Beato & Nistri1999, Cazalets et al. 1998, Cowley & Schmidt1995).
The nature of the inhibitory CPG net-works controlling the ipsilateral coordinationof flexors and extensors across the same or dif-ferent joints in a limb is for the most part un-known. Part of the rhythmic inhibition comesfrom commissural interneurons (CINs) (seeCircuits Involved in Left-Right Coordina-tion, below). However, an appropriate acti-vation between flexors and extensors is notdependent on crossing connections becauseit can persist in hemisected cords (Bonnotet al. 2002, Kato 1987, Kjaerulff & Kiehn1997). These findings demonstrate that ip-silateral inhibitory networks are strongly in-volved in flexor-extensor coordination. Fora long time Ia interneurons (Ia-INs) andRenshaw cells (RCs), two of the known in-hibitory spinal interneuron types projectingto motor neurons, have been implicated in this
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EN1: engrailed 1
Pax6: paired boxgene 6
coordination. Both types of cells are rhythmi-cally active during locomotion (McCrea et al.1980, Noga et al. 1987, Pratt & Jordan 1987).RCs fire in phase with the motor neurons theyreceive collaterals from and extensor Ia-INsfire out of phase with extensor motor neurons,whereas flexor Ia-INs fire out of phase withflexor motor neurons. Based on these find-ings it has been proposed that the importanceof RCs to the CPG is reserved to tuning thefiring rates of motor neurons and contributingto burst termination whereas Ia-INs are likelyinvolved in the rhythmic inhibition seen inmotor neurons during locomotion and there-fore are responsible for the flexor-extensor co-ordination (McCrea et al. 1980, Noga et al.1987, Pratt & Jordan 1987).
New knowledge from studies of neu-ronal populations expressing specific molecu-lar markers has, however, challenged this view(Gosgnach et al. 2006). Four different popu-lations of interneurons can be identified in theventral spinal cord based on their expressionpatterns of certain transcription factors. Theyare designated V0, V1, V2, and V3 neuronsand are marked by the expression of transcrip-tion factors Evx1/2 (even-skipped homeoboxgenes), engrailed 1 (En1), GATA2–3 (GATAbinding protein)/Chx10 (ceh-10 homeo do-main containing homolog), and Sim1 (single-minded homolog 1), respectively (for reviewsee Goulding et al. 2002, Goulding & Pfaff2005, Jessell 2000) (Figure 3). The develop-ment of the V1 population is controlled bypaired box gene 6 (Pax6), another transcrip-tion factor expressed upstream from En1 dur-ing embryonic development. En1-expressingneurons are inhibitory, locally and ipsilater-ally projecting, and give rise to RCs and Ia-INs (Biscoe et al. 1999, Eriscon et al. 1997,Sapir et al. 2004). In mice where Pax6 isknocked out, the number of V1 interneuronsis dramatically reduced (Sapir et al. 2004).These mice show slower walking and a lessabrupt termination of the motor bursting(Gosgnach et al. 2006) (Figure 4). A sim-ilar phenotype is found when V1 neurons
are selectively killed by driving the expres-sion of diphteria toxin A (DTA) by the En1promotor (Figure 4). This suggests V1 in-terneurons (and thereby RCs and Ia-INs) areinvolved in determining burst shape and dura-tion. However, flexor-extensor coordinationis preserved in Pax6−/− and En1-DTA mice,even in the hemicord, and motor neuronsstill receive rhythmic inhibition. These ex-periments show that even when the RCs, Ia-INs, and crossed inhibitory input are greatlyreduced or absent, it is still possible to pro-duce an alternating flexor-extensor rhythm,suggesting the existence of local, ipsilateral in-hibitory interneurons not yet identified as re-sponsible for the flexor-extensor alternation.A major aim of future studies should there-fore be to identify this population of interneu-rons directly involved in flexor-extensor coor-dination in the mammalian locomotor CPG,a population of cells that that has no coun-terpart in the lamprey, tadpole, or zebrafishswimming CPGs.
The step cycle was dramatically increasedin Pax6−/− and En1-DTA mice (Figure 4)as compared with controls. This surpris-ing finding indicates that the ipsilateralinhibitory network belonging to the V1population is involved in setting the locomo-tor frequency, possibly by a feedback mecha-nism to the rhythm-generating network. Be-cause RCs and Ia-INs do not project to anyother interneurons besides other RCs and Ia-INs, unidentified V1 interneurons must beinvolved in this feedback. The exact mech-anism for the slowing effect is at the momentunclear. However, En1-positive interneuronsin tadpole and zebrafish recently have beenfound to have direct connections to excita-tory and commissural CPG neurons, as well asmotor neurons and sensory interneurons. Intadpole and zebrafish En1-positive cells makeup all ipsilaterally projecting inhibitory in-terneurons and form a morphologically andphysiologically homogeneous population ofneurons (Higashijima et al. 2004, Li et al.2004). In these animals the En1 neurons are
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Figure 3Summary diagram of developmental expression of transcription factors in mammalian spinalinterneurons. V0 interneurons (INs) express Evx1; V1 INs, En1; V2 INs, Chx10 or Gata2/3; and V3 INs,Sim1. Motor neurons express HB9. These neuronal populations develop from progenitor cellpopulations (p0, p1, p2, p3, and pMN) located in the ventricular zone. The projecting patterns of thepostmitotic neuron populations are depicted in the figure. Adapted from Jessell (2000) and Goulding &Pfaff (2005). FP, floor plate; RP, roof plate.
not responsible for the midcycle inhibitionunderlying left-right alternation but preventsustained firing by providing early rhyth-mic inhibition. Removing such an inhibi-tion is likely to cause longer bursting in allCPG neurons and impose a slowing effecton the step period. Although En1 neuronsclearly do not constitute a homogeneous pop-ulation of neurons in mammals, En1 neu-rons as a meta-class of neurons may havepreserved some if not all of their phyloge-netic older characteristics in mammals butnow may be subdivided into several neuronalpopulations.
CIRCUITS INVOLVED INLEFT-RIGHT COORDINATION
Muscle activity on the left and right side ofthe body is precisely coordinated during loco-motion to secure alternation of correspondingmuscles on either side of the body (e.g., hipflexors) as seen during walking or synchronyas seen during galloping. The neuronal cir-cuits responsible for left-right coordinationare CINs whose axons cross the midline viathe ventral commissure. These neurons haverecently been studied extensively in the ro-dent and cat spinal cord partly because theycan be uniquely identified anatomically and
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Figure 4Deletion of V1 neurons leads to slow walking. Recordings of drug-induced (5-HT plus NMDA)locomotor-like activity in E18.5 spinal cords from wild-type (control), Pax6−/−/, and En1-DTA mice.The circular plots show that the left-right (lL2-rL2) and flexor-extensor alternation (lL2-lL5) are normalin both Pax6−/− and En1-DTA mice. The speed of locomotion is, however, dramatically reduced in themutants as compared with wild type. Adapted from Gosgnach et al. (2006) (with permission from NaturePublishing Group, copyright 2006).
physiologically because they project axonsacross the midline.
Anatomical Organization
The basic organization of CINs in thehindlimb region of the spinal cord has been re-vealed by lesioning studies that have demon-strated left-right coordination is mediated byventrally located CINs (Kjaerulff & Kiehn1996) and that the left-right coordinationis distributed over extended regions of thehindlimb spinal cord (Cowley & Schmidt1997, Kjaerulff & Kiehn 1996, Kremer &
Lev-Tov 1997). Anatomical tracing studiesand intracellular staining studies have shownthat CINs in the ventral mammalian spinalcord (laminae VII, VIII, and X) can be sub-divided into two major categories based ontheir axonal projection: intrasegmental andintersegmental CINs. Intersegmental CINshave long axons that project at least two seg-ments. They can be subdivided into (a) as-cending CINs, (b) descending CINs, and (c)bifurcating CINs (Bannatyne et al. 2003, Eideet al. 1999, Hoover & Durkovic 1992, Mat-suyama et al. 2004, Nakayama et al. 2002,Nissen et al. 2005, Stokke et al. 2002). It is
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plausible these anatomically defined groupsof cells serve distinct roles in the locomotorcircuits and they are therefore considered inseparate sections below.
Activity in Descending CommissuralInterneurons are Involved in BindingSynergies Across the Cord
Descending CINs (dCINs) located in the ven-tral horn in lumbar segment L2 of the spinalcord and which have axons projecting con-tralaterally at least as far as lumbar segmentL4 have been studied extensively in the ro-dent (Butt et al. 2002a, 2002b; Butt & Kiehn2003). Three-quarters of these neurons showrhythmic activity with the locomotor pattern.In contrast to what is found in the lampreyand tadpole, these dCINs fired at all phasesof the cycle, both in phase and out of phasewith ipsilateral flexor motor neurons. Analy-sis of the synaptic effects on L4 motor neu-rons revealed that L2 dCINs are composedof a mixed population of glutamatergic andglycinergic neurons, many of which terminatedirectly onto flexor and extensor motor neu-rons in L4. When comparing the phase of fir-ing with the synaptic effect on motor neurons,it was predicted that the L2 dCINs serve arole in providing appropriate drive to flexor-and extensor-timed motor neuron activity inmore caudal spinal segments. Thus, dCINsthat fire in phase with ipsilateral flexors canexcite contralateral extensor motor neuronsvia a glutamatergic pathway, whereas othersinhibit contralateral flexor motor neurons viaa glycinergic pathway (Figure 5). dCINs thatfire in the extensor phase inhibit contralat-eral extensors and excite contralateral flexors.These findings indicate that L2 dCINs assistin binding synergies across the cord to pro-vide the appropriate crossed muscle coordina-tion during locomotion. Although the dCINsmake up a heterogeneous group of neurons,their activity is coordinated as a functionalunit. These experiments show that by care-fully relating the firing pattern and the post-synaptic effects, it is possible to deduce the
Ipsi-L2flexor
Contra-L4extensor
iL2 CIN
Ipsi-L2extensor
Contra-L4flexor
iL2 CIN
Ipsi-L2flexor
Contra-L4flexor
iL2 CIN
Ipsi-L2extensor
Contra-L4extensor
iL2 CIN
Excitatory Inhibitory
Figure 5Functional role of the descending commissural interneurons (dCINs) in themammalian locomotor network. dCINs are either excitatory (left panels) orinhibitory (right panels). The excitatory and inhibitory dCINs fire in phasewith both ipsilateral L2 flexors and ipsilateral L2 extensors. These differentcategories of dCINs therefore bind ipsilateral L2-flexor/contralateralL4-extensor and ipsilateral L2-extensor/contralateral L4-flexor synergiesacross the cord with excitation and ipsilateral L2-flexor/contralateralL4-flexor and ipsilateral L2-extensor/contralateral L4-extensor antagonistswith inhibition. Adapted from Kiehn & Butt (2003) and Butt & Kiehn(2003) (with permission from Elsevier, copyright 2003).
role of a group of rhythmically active in-terneurons in the CPG.
Populations of dCINs located in the CPGregion of the cat (lamina VIII) show manysimilarities to those described in the rodent.These dCINs have somata located in L3–L5in the cat (corresponding to Th13–L2 in therodent) and axons projecting to extensor mo-tor neurons in L7 (corresponding to L4/L5in the rodent) (Bannatyne et al. 2003; Edgleyet al. 2003; Hammar et al. 2004; Jankowskaet al. 2003, 2005a, 2005b; Krutki et al. 2003;Matsuyama & Jankowska 2004). They appearto provide monosynaptic glutamatergic orglycinergic, as well as polysynaptic, inhibitoryinputs to caudal motor neurons (Bannatyneet al. 2003, Jankowska et al. 2003). The ax-onal projections of the dCINs are widespreadover many segments and often have arboriza-tions both within the motor neuron poolsand in the intermediate area where puta-tive CPG interneurons are located, suggesting
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these dCINs not only coordinate motor neu-ron activity, but also provide direct input tointerneurons on the contralateral side (Ban-natyne et al. 2003, Matsuyama et al. 2004).Although it has not been shown directly thatthese midlumbar dCINs were rhythmicallyactive during locomotion, suggestive evidencecomes from the fact that these cells receivemonosynaptic input from large reticulospinalinterneurons that in turn are active during lo-comotion. In a separate study, however, Mat-suyama et al. (2004) showed that all dCINslocated in L4–L6 of the cat are rhythmicallyactive during locomotion evoked by stimu-lation of brainstem and midbrain locomo-tor regions. All together this suggests thedCINs in the cat may be functionally orga-nized in a similar way to that described inrodents.
Role of Ascending CommissuralInterneurons
Rhythmic, ascending lumbar CINs, some ofwhich are cholinergic, have been describedboth in the cat (Huang et al. 2000, Mat-suyama et al. 2004) and the rodent (Carlinet al. 2006, Zhong et al. 2006). Many of theseneurons have long, ascending fibers and mighttherefore, in addition to being involved inhindlimb coordination, provide rhythmic sig-nals to the forelimb region of the spinal cordor the brainstem. Indeed recent experimentsshow that the hindlimb CPG provides strongrhythmic inputs to the forelimb CPG and thatpart of this drive is crossed inhibition (Juvinet al. 2005).
Ascending CINs have also been classi-fied by their expression pattern of transcrip-tion factors. Of the four metaclasses of in-terneurons characterized in this way, two ofthem, V0 and V3 interneurons, include CINs(Goulding & Pfaff 2005, Goulding et al. 2002,Moran-Rivard et al. 2001, Pierani et al. 2001).V0 interneurons constitute a group of pureCINs with ascending axons, whereas V3 in-terneurons constitute a mixed population ofCINs and ipsilaterally projecting interneu-
rons. The V0 population can be ablated fromthe cord by knocking out the fate-determiningtranscription factor Dbx (developing brainhomeobox) that is expressed in progenitors toV0 interneurons (Figure 6a) (Lanuza et al.2004; Pierani et al. 1999, 2001). These in-terneurons make monosynaptic connectionsto contralateral motor neurons (Figure 6b).When ablated (Figure 6b) there is a change inthe left-right coordination (Figure 6d ), withperiods of synchrony intermingled with nor-mal alternation. This observation led Lanuzaet al. (2004) to suggest that V0 interneuronsare directly involved in the left-right alterna-tion during locomotion. That the left-rightalternation was not completely replaced byleft-right synchrony indicates that other CINsbelonging to a non-V0 group of interneuronsare involved in left-right alternation.
Functional Organization ofIntrasegmental CommissuralInterneurons
The intrasegmental connections are likely toplay a direct role in organizing the left-rightcoordination between segmental, homony-mous muscles. Studies using a longitudinalsplit-bath preparation in the rodent suggesta substantial proportion of the crossed in-trasegmental coordination during alternatingactivity is mediated via glutamatergic CINsthat act on inhibitory interneurons located ip-silateral to the motor neurons (Kjaerulff &Kiehn 1997). Possible candidates for these re-lay interneurons are Ia-INs and RCs. In ad-dition to this indirect effect, there also ap-pears to be direct inhibitory connections fromCINs onto motor neurons during alternat-ing activity. Direct support for this networkconnectivity comes from studies in mice andthe cat that show that Ia-IN and RCs canbe excited directly by CINs (Jankowska et al.2005b; H. Nishimaru, C.E. Restrepo & O.Kiehn, manuscript submitted) and that in-trasegmental CINs in mice can provide bothmono- and polysynaptic inhibition of motorneurons (Quinlan & Kiehn 2005) (Figure 7).
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Figure 6Deletion of the spinal V0 commissural interneuron population leads to increased left-right synchronyduring locomotion. (a) Summary of changes in V0 cell fate in the Dbx1 mutant spinal cord. Knockout ofthe Dbx1 gene causes a loss of the V0 population that is composed of V0D (dorsally located) Evx1negative neurons and V0V (ventrally located) Evx1 positive neurons. The V1 loss is accompanied by anincrease in the V1 population (ipsilaterally projecting inhibitory neurons) and the dorsally located dI6population, which is also comissurally projecting. dI6, dorsal interneurons 6; Lbx, ladybird familyhomeobox. (b) V0 interneurons make synapses with contralaterally located motor neurons (MN) asshown by trans-synaptic transport of pseudorabies virus (PRV-GFP) from motor neurons to interneurons36 or 48 h after injection of PRV into the muscle. V0 interneurons are shown in yellow, whereascommissural interneurons that were non-V0 are shown in green. (c and d) Locomotor activity in Dbx1mutant mice. Schematic of recording setup with recordings of activity from left and right L2 (lL2, rL2)and from left L5 (lL5) ventral roots. Locomotor activity induced by a combination of NMDA and 5-HT.Appropriate left-right alternation (lL2 and rL2) is observed in wild-type mice as is flexor (lL2) andextensor (lL5) alternation. This is seen as phase values of approximately 0.5 in the circular plots to theright. In the Dbx1 mutant the locomotor activity exhibits periods of synchronous activity in the lL2 andrL2 ventral roots as well as alternation whereas the flexor-extensor alternation appears normal. Thedisturbances in left alternations in Dbx1 mutants are seen as two clusters of phase values in the circularplot of lL2–rL2, one at approximately 0.5 representing alternation and a smaller one at approximately 1.0representing synchronicity. Data in panel a adapted from Pierani et al. (2001) and Lanuza et al. (2004).All other data adapted from Lanuza et al. (2004) (with permission from Elsevier, copyright 2004).
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CIN
CIN
CIN
Ia
RC
MN
Midline Descending
Figure 7Summary diagram of intrasegmental commissuralinterneuron (CIN) connections. Excitatorysynapses are represented by open circles, whereasinhibitory synapses are represented by closedcircles. MN, motor neuron.
In addition to inhibitory pathways, directexcitatory pathways are also found betweenintrasegmental CINs (sCINs) and segmen-tal motor neurons on the contralateral side(Quinlan & Kiehn 2005) (Figure 7). Theemerging picture from these findings is thatthere is a dual intrasegmental CIN systemthat inhibits segmental motor neurons via(a) polysynaptic inhibition mediated by glu-tamatergic sCINs and local ipsilaterally pro-jecting inhibitory interneurons including RCsand Ia-INs and (b) monosynaptic inhibitionmediated via glycinergic/GABAergic sCINs.In addition to the inhibitory pathways, glu-tamatergic sCINs can excite motor neuronsdirectly. The CIN circuitries found in mam-mals are thus much more complex thanthose described in lamprey and tadpole wherelocomotor-related CINs are glycinergic andmonosynaptically connected to neurons onthe contralateral side.
Although the segmental CIN connectionsonto motor neurons will contribute directly totheir activity, they are obviously not respon-sible for the precise coordination of rhythmicactivity on the two sides of the cord. For thisto happen, CINs need to connect to rhythm-generating CPG elements on the other side
of the cord. In the lamprey and tadpole, in-hibitory CINs connect to excitatory interneu-rons, local inhibitory interneurons, and con-tralateral CINs as well as motor neurons onthe other side of the cord. It is the direct inhi-bition of excitatory interneurons (the rhythm-generating core) that is thought to cause theswitch from activity to silence. Because therhythm-generating core has not been de-fined in mammals, it is unknown if a similarconnectivity pattern is present in mammals.Some phylogenetic conservatism seems, how-ever, to be in place because CINs in mam-mals, like in lamprey and tadpole, are recipro-cally connected across the cord (Birinyi et al.2003).
Another important issue related to the in-trasegmental CINs is what happens when thecoordination is changed from alternation tosynchrony. One possibility is that the crosseddual inhibitory systems are active during walk-ing, whereas the excitatory systems are activeduring segmental synchronous activity suchas hopping or gallop. These two systems arethen turned on and off during the differentpatterns of activity. Another possibility is thatgait changes happen because of a slight changein the balance between inhibitory and excita-tory crossed actions. This implies both sys-tems are active simultaneously, but one orthe other system dominates depending on thegait. The latter hypothesis is made conceiv-able by experiments in knockout mice with anabnormal increase in crossed excitation thatleads to a hopping gait (Kullander et al. 2003).In these experiments the right-left synchronycould be switched into alternation by phar-macologically increasing the drive in the nor-mal inhibitory CIN system, thereby chang-ing the balance between crossed excitationand inhibition. How such a change of balancecould take place during normal gait changesis, of course, unknown. One possibility is thatdescending modulatory fibers have specificinhibitory and/or excitatory actions on thecrossed inhibitory and excitatory CIN path-ways (Hammar et al. 2004).
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Commissural Interneurons andRhythm Generation
Are CINs involved in rhythmogenesis inmammals? Clearly they are not the onlysource because the rhythm can be inducedin the hemicord both in rodents (Bonnotet al. 2002, Kjaerulff & Kiehn 1997, Kudo& Yamada 1987, Nishimaru et al. 2006) andthe cat (Kato 1987). However, when only oneside of the cord is active, the locomotor periodis significantly longer while the burst ampli-tude is unchanged (Kjaerulff & Kiehn 1997).This observation strongly suggests CINs havea rhythmogenic role in the mammalian loco-motor CPG in addition to their role in coor-dinating the left-right activity, similar to whathas been observed in the turtle (Stein et al.1995).
GENETIC NETWORKCRACKING
The large numbers of cells present in themammalian spinal cord place major con-straints on electrophysiological network anal-yses of the locomotor CPG. Although neu-rons belonging to a certain population are notrandomly distributed in the ventral horn, theyare always intermingled with other cell pop-ulations, which makes it a tedious task to re-peatedly record from identified populationsof neurons and characterize new ones. Label-ing of neurons with genetically encoded re-porters such as green fluorescent protein orbeta-galactosidase expressed under the con-trol of specific promoters has therefore provento be a powerful tool for CPG network anal-ysis in mice (Butt et al. 2005, Hinckley et al.2005, Wilson et al. 2005; see also Fetcho &Bhatt 2004). Electrophysiological techniques,although needed to reveal functional connec-tivity and cellular properties, also fail to estab-lish causality between the activity in a group ofneurons and its function in the CPG. For ex-ample, although we can deduce the functionof the different groups of CINs from theirfiring properties and functional connectivity,
AlstR: allatostatinG-protein coupledreceptor
this analysis will never allow us to determineif these populations are necessary or sufficientto encode a particular aspect of the locomo-tor behavior. Such knowledge can only beobtained from functional gain or loss exper-iments by selectively activating or inactivat-ing specific groups of neurons in the network.Functional loss studies using pharmacologicalmanipulation of cellular properties (Ramirezet al. 2004) or selective killing of neuronswith substance-P-saporin (Gray et al. 2001)have been used in the respiratory CPG. An-other approach is to use genetically controlledmanipulations, where the manipulations areperformed in a cell-specific manner. Thesemanipulations include cell knockout andelectrically silencing cells, specific block ofsynaptic release, or photochemical activa-tion (for details and discussion of tech-nical/experimental difficulties, see Callaway2005, Kiehn & Butt 2003, Kiehn & Kullan-der 2004, Miesenbock & Kevrekidis 2005).Knockout of genetically defined populationsof spinal interneurons has, as described above,already given important information aboutthe contribution of groups of interneurons tothe generation and/or coordination of the mo-tor output (Gosgnach et al. 2006, Lanuza et al.2004). Chronic perturbations of the networkmight, however, cause activity-dependent re-organization of the CPG (Myers et al. 2005).
An important addition to the chronic ge-netic manipulations is, therefore, the abilityto inactivate or activate the cell populationsacutely and reversibly. Such acute, reversiblemethods have now been developed bothfor activation (Lima & Miesenbock 2005,Miesenbock & Kevrekidis 2005) and forinactivation of neuronal activity (Callaway2005). One of the acute inactivation methodsis the cell-specific expression of the Drosophilaallatostatin G protein–coupled receptor(AlstR), which is not found normally in themammalian central nervous system and, whenactivated by its agonist, activates an inwardrectifier and thereby decreases neuronalexcitability (Callaway 2005, Lechner et al.2002). This versatile method has recently
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Figure 8(a) Experiments with expression of the allatostatin (AL) receptor in V1 neurons show that acute silencingof V1 neurons via activation of the allatostatin receptor causes a slowing of locomotor-like rhythmicitysimilar to what is observed in the Pax6−/− and En1-DTA mice. Adapted from Gosgnach et al. (2006)(with permission from Nature Publishing Group, copyright 2006).
been used in locomotor experiments to ex-press the AlstR selectively in inhibitory V1 in-terneurons (Gosgnach et al. 2006). Acute ac-tivation of the AlstR in V1 neurons markedlylengthened the step cycle similar to what wasseen when V1 neurons where chronicallyinactivated in the Pax6−/− EN1-DTA mice(Figure 8). These types of experimentsdemonstrate that the acute silencing of aselect population of neurons using geneticapproaches can be used to elucidate theirfunction with respect to a defined behavior.The silencing techniques therefore providepowerful tools to dissect the network. It willbe advantageous, however, to combine themwith genetically driven activation techniques.For example if the selective activation of agroup of excitatory CPG neurons leads torhythmic motor outputs, it would stronglysuggest these cells are directly involved inrhythm generation (see Role in RhythmGeneration, above).
Despite the promising role for genetic ap-proaches to dissect neuronal networks andtechnical problems notwithstanding, there arealso reasons to be cautious. In its ideal versionthe genetic approach is based on the assump-tion that functional groups of spinal interneu-rons can be uniquely characterized by the ex-pression of one or a few molecular markers.
In reality none of the published genetic mark-ers are completely specific to interneurons(HB9), nor do they delineate an entirely ho-mogenous group of interneurons (transcrip-tion factors expressed in V0–V3 interneuronsor EphA4). Results from genetic manipula-tion of a mixed population of interneurons,therefore, need to be interpreted carefully,and more selective markers for functionalpopulations of neurons are strongly needed.Thus selective molecular markers for electro-physiologically defined populations of puta-tive CPG neurons such as the Ia-INs, RCs,excitatory EphA4 neurons, or intrasegmentalinhibitory and excitatory CINs would be oftremendous value. It is possible such markerswill appear as a result of large genetic screen-ing of neuronal markers (Gong et al. 2003,Gray et al. 2004). With luck such screeningprocedures could also reveal specific expres-sion patterns of molecular markers that mightprovide the basis for the identification of newpopulations of mammalian locomotor CPGinterneurons.
SUMMARY AND PERSPECTIVES
Over the past decade there has been a rapidadvance in our knowledge of the mammalianlocomotor network. Important aspects of the
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overall network structure have been revealedfrom lesioning and pharmacological studies.Together these studies strongly indicate thatthe mammalian locomotor CPG is distributedalong the spinal cord, with a rostral-caudalgradient in rhythmogenic potential. Pharma-cological experiments also suggest that therhythmogenic core of the network is com-posed of excitatory, ipsilaterally projecting in-terneurons. Although these cells have not beidentified with certainty, rhythmically active,non-overlapping populations of excitatory in-terneurons (which are candidate neurons toprovide direct or indirect rhythmic input toipsilateral motor neurons during locomotion,and thus could be generating the rhythm)have been localized both in the cat and themouse spinal cord. The evidence that thesecells are involved in rhythm generation is indi-rect, however, and future experiments shouldtherefore aim at developing inactivation oractivation experiments that can directly anddefinitively define neurons as members of theexcitatory core circuitry, determine how thesecircuits generate rhythmic bursting, how theyare coupled along the cord, and how ipsilat-erally projecting inhibitory neurons are con-nected to the excitatory core circuitry and howthey are playing a role in setting the speed oflocomotion.
In addition to the rhythm-generating ex-citatory core, two other elements in the net-work can be isolated: flexor and extensorcoordinating circuits and left-right coordinat-ing circuits. Knowledge about the extensor-flexor coordination circuitries is at themoment rudimentary. Known ipsilaterallyprojecting inhibitory interneurons, such asRCs and Ia-INs, may contribute to rhythmicmotor neuron inhibition but genetic knock-out and silencing experiments indicate thatalthough these inhibitory cell populations aredispensable for flexor-extensor coordination,they are involved in speed regulation. A ma-jor aim of future studies should therefore beto identify the population of interneurons di-rectly involved in flexor-extensor coordina-tion in the mammalian walking CPG.
The left-right coordinating circuitries arethose network circuitries best understood inthe mammalian walking CPG. Anatomicaland electrophysiological analyses both in thecat and rodent have shown that these com-plex circuitries are composed of intrasegmen-tal and intersegmental CINs that are both ex-citatory and inhibitory. A functional analysisof these circuitries suggests that intrasegmen-tal CINs are involved in binding motor syn-ergies along the cord, whereas intersegmen-tal CINs appear to be directly involved in thecoordination of homonymous muscle activ-ity at a segmental level. These studies clearlydemonstrate that the mammalian CPG con-nectivity pattern is complex but that it can berevealed to a level where basic understandingof the network component can be deduced.
Genetic approaches are an important newaddition to the classical electrophysiologi-cal network analysis in mammals. These ap-proaches can potentially directly link a popu-lation of interneurons to a network functionby selectively silencing or ablating the popula-tion from the network. Such experiments haverecently been applied to the mouse spinal cordand have given new insights to the functionof identified populations of spinal interneu-rons, like the En1 ipsilaterally projecting in-hibitory interneurons. The power of these ex-periments is obvious. However, to be fullysuccessful the experiments need to be appliedto more specific, homogenous neuronal pop-ulations than those defined by the presentlyavailable molecular markers. For this to hap-pen a recursive process involving molecu-lar, anatomical, and electrophysiological ap-proaches is needed.
When comparing what is known about themammalian walking CPG with the much bet-ter understood vertebrate swimming CPGs, itis clear that some elementary features of thevertebrate spinal locomotor network struc-ture are preserved phylogenetically. Notably,the CPG network is distributed and includesexcitatory neurons that are responsible forrhythm generation and glycinergic CINs thatare directly involved in left-right alternation.
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These basic network elements outline gen-eral principles of organization and functionin vertebrate locomotor circuitries. Notwith-standing these similarities in network designthere are also significant differences. New net-work elements appear to be added to the CPGnetwork when moving from a nonlimbed to alimbed animal. These elements include CINcircuitries that are more complex segmen-tally than in the swimming CPG, as wellas CIN circuitries that bind motor synergiesacross limbs and ipsilateral inhibitory net-works involved in flexor-extensor coordina-
tion. Moreover, whereas network elements inthe swimming CPG appear to be composed ofhomogenous populations of neurons, similarnetwork elements (e.g., the excitatory core) inthe walking CPG appears to be composed ofmore heterogeneous populations of neurons.The added network complexity that makes thewalking CPG significantly different from theswimming CPG is probably a reflection of thehigh flexibility in mammalian locomotion thatallows for the production of many differentgaits and their functional adaptation acrosschanging environments.
ACKNOWLEDGMENTS
The work in Kiehn’s lab is supported by the Human Frontier Science Program, NIH, TheKarolinska Institute, and the Swedish Research Council. I thank my colleagues for many in-spiring discussions regarding the central issues raised in this review.
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Contents ARI 25 May 2006 20:33
Annual Reviewof Neuroscience
Volume 29, 2006Contents
Adaptive Roles of Programmed Cell Death During Nervous SystemDevelopmentRobert R. Buss, Woong Sun, and Ronald W. Oppenheim � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1
Endocannabinoid-Mediated Synaptic Plasticity in the CNSVivien Chevaleyre, Kanji A. Takahashi, and Pablo E. Castillo � � � � � � � � � � � � � � � � � � � � � � � � � � � � �37
Noncoding RNAs in the Mammalian Central Nervous SystemXinwei Cao, Gene Yeo, Alysson R. Muotri, Tomoko Kuwabara,and Fred H. Gage � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �77
The Organization of Behavioral Repertoire in Motor CortexMichael Graziano � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 105
TRP Ion Channels and Temperature SensationAjay Dhaka, Veena Viswanath, and Ardem Patapoutian � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 135
Early Events in Olfactory ProcessingRachel I. Wilson and Zachary F. Mainen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 163
Cortical Algorithms for Perceptual GroupingPieter R. Roelfsema � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 203
Deep Brain StimulationJoel S. Perlmutter and Jonathan W. Mink � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 229
RNA-Mediated Neuromuscular DisordersLaura P.W. Ranum and Thomas A. Cooper � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 259
Locomotor Circuits in the Mammalian Spinal CordOle Kiehn � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 279
Homeostatic Control of Neural Activity: From Phenomenology toMolecular DesignGraeme W. Davis � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 307
Organelles and Trafficking Machinery for Postsynaptic PlasticityMatthew J. Kennedy and Michael D. Ehlers � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 325
Noncanonical Wnt Signaling and Neural PolarityMireille Montcouquiol, E. Bryan Crenshaw, III, and Matthew W. Kelley � � � � � � � � � � � � � � � 363
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Contents ARI 25 May 2006 20:33
Pathomechanisms in Channelopathies of Skeletal Muscle and BrainStephen C. Cannon � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 387
Imaging Valuation Models in Human ChoiceP. Read Montague, Brooks King-Casas, and Jonathan D. Cohen � � � � � � � � � � � � � � � � � � � � � � � � � 417
Brain Work and Brain ImagingMarcus E. Raichle and Mark A. Mintun � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 449
Complete Functional Characterization of Sensory Neurons by SystemIdentificationMichael C.-K. Wu, Stephen V. David, and Jack L. Gallant � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 477
Neurotrophins: Mediators and Modulators of PainSophie Pezet and Stephen B. McMahon � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 507
The Hedgehog Pathway and Neurological DisordersTammy Dellovade, Justyna T. Romer, Tom Curran, and Lee L. Rubin � � � � � � � � � � � � � � � � � � 539
Neural Mechanisms of Addiction: The Role of Reward-RelatedLearning and MemorySteven E. Hyman, Robert C. Malenka, and Eric J. Nestler � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 565
INDEXES
Subject Index � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 599
Cumulative Index of Contributing Authors, Volumes 20–29 � � � � � � � � � � � � � � � � � � � � � � � � � � � 613
Cumulative Index of Chapter Titles, Volumes 20–29 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 617
ERRATA
An online log of corrections to Annual Review of Neuroscience chapters (if any, 1977 tothe present) may be found at http://neuro.annualreviews.org/
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