Neuron

ArticleV1 and V2b Interneurons Secure the Alternating
Flexor-Extensor Motor Activity Mice Requirefor Limbed Locomotion
Jingming Zhang,1,6 Guillermo M. Lanuza,1,2,6 Olivier Britz,1 Zhi Wang,3 Valerie C. Siembab,4 Ying Zhang,1,7
Tomoko Velasquez,1 Francisco J. Alvarez,4,5 Eric Frank,3 and Martyn Goulding1,*
1Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA
2Fundación Instituto Leloir, IIBBA-CONICET, Buenos Aires 1405, Argentina
3Department of Molecular Physiology and Pharmacology, Tufts University School of Medicine, Boston, MA 02111, USA
4Department of Neurosciences, Cell Biology, and Physiology, Wright State University, Dayton, OH 45435, USA
5Department of Physiology, Emory University, Atlanta, GA 30322, USA
6Co-first author
7Present address: Department of Anatomy and Neurobiology, Dalhousie University, Halifax, NS B3H 4R2, Canada*Correspondence: goulding@salk.edu
http://dx.doi.org/10.1016/j.neuron.2014.02.013

SUMMARY

Reciprocal activation of flexor and extensor muscles constitutes the fundamental mechanism that
tetrapod vertebrates use for locomotion and limbdriven reflex behaviors. This aspect of motor coordination is controlled by inhibitory neurons in the spinal
cord; however, the identity of the spinal interneurons
that serve this function is not known. Here, we showthat the production of an alternating flexor-extensor
motor rhythm depends on the composite activitiesof two classes of ventrally located inhibitory neurons,
V1 and V2b interneurons (INs). Abrogating V1 andV2b IN-derived neurotransmission in the isolated
spinal cord results in a synchronous pattern of L2
flexor-related and L5 extensor-related locomotor
activity. Mice lacking V1 and V2b inhibition are unable to articulate their limb joints and display marked
deficits in limb-driven reflex movements. Taken
together, these findings identify V1- and V2b-derived
neurons as the core interneuronal components of
the limb central pattern generator (CPG) that coordinate flexor-extensor motor activity.

INTRODUCTION
Terrestrial vertebrates use their limbs for a range of motor tasks,
from simple protective reflexes and locomotion to more complex
volitional movements, such as reaching, grasping, and grooming. These motor behaviors require the production of a reciprocating pattern of motor impulses to antagonist groups of flexorand extensor muscles (Sherrington, 1893; Grillner, 1975).
Multiple studies have shown that flexor-extensor alternation
is an intrinsic property of the locomotor central pattern generator(CPG) in limbed animals (Brown, 1911; Eccles et al., 1956;
Goulding, 2009; Grillner, 1975; Grillner and Jessell, 2009; Kiehn,
138 Neuron 82, 138–150, April 2, 2014 ª2014 Elsevier Inc.

2006; Ladle et al., 2007). However, efforts to identify the interneuron (IN) cell types that secure flexor-extensor alternation
have met with limited success, and because of this we stillknow very little about the overall organization of the locomotor
CPG in limbed vertebrates.
Prior efforts to interrogate the structure of the neural networks
that control flexor-extensor alternation have shown that the
flexor-extensor control system is composed of inhibitory neurons that reside in each half of the spinal cord (Cowley andSchmidt, 1995; Sernagor et al., 1995; Talpalar et al., 2011; Whelan et al., 2000). A major drawback of the pharmacological approaches used in these studies is the widespread inactivationof inhibitory neurons regardless of their subtype or connectivity(Cowley and Schmidt, 1995; Bracci et al., 1996; Kremer andLev-Tov, 1997; Cazalets et al., 1998). This has precluded amore detailed determination of the neuronal cell types limbed
animals use to produce an alternating flexor-extensor motor
rhythm. More recently, genetic approaches in mice that selectively inactivate or delete specific interneuron classes havebeen employed to determine the contribution molecularly
defined classes of INs make to locomotion (Crone et al., 2008;
Gosgnach et al., 2006; Lanuza et al., 2004; Zhang et al., 2008;
Zagoraiou et al., 2009). Whereas these functional studies haveidentified neurons with selective roles in regulating left-right
coordination, rhythmogenesis, and the speed of the step cycle,
the cells that are responsible for establishing an alternating
flexor-extensor rhythm have still not been isolated (Goulding,2009; Grillner and Jessell, 2009; Kiehn, 2006; Stepien and Arber,2008).
Initial attempts to determine the molecular identity anddevelopmental provenance of the spinal INs that establish thealternating flexor-extensor motor activity mice use for limb
movements focused on V1 INs. V1 INs are a class of ipsilaterally projecting inhibitory neurons (Betley et al., 2009; Sapir
et al., 2004; Saueressig et al., 1999) that includes cells possessing the anatomical features of reciprocal Ia inhibitory interneurons (IaINs; Alvarez et al., 2005), a cell type thought to playa prominent role in flexor-extensor inhibition (Eccles et al.,1956; Feldman and Orlovsky, 1975). However, spinal cords

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Neuronal Control of Flexor-Extensor Alternation

Figure 1. A Non-V1 IN Population
Is Involved in Securing Reciprocal FlexorExtensor Activity(A and B) Left: Representative extracellular recordings from ventral roots of a P0 En1Cre;
R26floxstop-TeNT spinal cord showing locomotor
ENG activity prior to and after hemisection. Signals were recorded from the L2, iL5, and cL2
ventral roots. Right: Circular plots showing averaged L2-iL5 phase relationships in whole (upper)and hemisected (lower) preparations. Inner circle
indicates an r significance value of p = 0.01. Points
located near 0.5 represent alternation. Alternating
L2-iL5 locomotor activity is maintained before andafter hemisection. The L2-cL2 phase relationshipis also shown. The number of cords analyzed isindicated (n).
(C and D) Representative sections of FITCdextran-injected spinal cords from wild-type andPax6 / embryos at E18.5, showing FITCdextran+ cells rostral and ipsilateral to the application site. vGAT in situ hybridization identifies a
subset of FITC-dextran+ cells as inhibitory neurons
in wild-type and Pax6 / spinal cords (arrowheads).
(E) A section from an FITC-dextran-labeled E18.5
Pax6 / embryo showing FITC-dextran+/Gata3+
cells (yellow, arrowheads).
(F–H) Comparison of Gata3+ V2b cell numbers inE11.5 Gata3lacZ and Gata3lacZ; Pax6 / spinal
cords as assessed by b-galactosidase staining.
Quantification is shown in (H) (mean ± SEM; n = 12
sections, three cords for each sample).

RESULTSlacking V1 INs retain reciprocal Ia inhibition (Wang et al., 2008),
and they produce an alternating pattern of flexor-extensor
locomotor activity (Gosgnach et al., 2006). We now show that
V2b INs cooperate with V1 INs to secure the alternating patternof flexor-extensor motor activity that is necessary for limbed
locomotion. We also find that cells with the characteristic
properties of IaINs develop from both V1 and V2b INs. Taken
together, our results demonstrate that flexor-extensor control
is a distributed property of the walking CPG shared by V1
and V2b IN cell types. Interestingly, V1 and V2b INs share acommon phylogenetic heritage with two classes of inhibitory
neurons in the spinal cords of aquatic vertebrates. This suggests that the neurons walking vertebrates employ for flexorextensor control were originally part of the swimming CPG
and were recruited for this new function during the course ofevolution.

A Population of Non-V1 INs Secure
Alternating Flexor-Extensor
Activity
As a first step toward defining the cellularorganization of the circuits that secure
flexor-extensor alternation in mice, we
examined the pattern of motor activity inhemisected spinal cords lacking V1 INderived inhibition. Severing all commissural connections in the
En1Cre; R26floxstop-TeNT spinal cord produced a strictly reciprocating L2 (flexor-related) and iL5 (ipsilateral extensor-related)motor rhythm (Figure 1B; n = 11 hemicords) when locomotorlike activity was induced with N-methyl-D aspartic acid
(NMDA) and 5-hydroytryptamine (5-HT). This pattern of
L2-iL5 activity was similar to that seen in the intact En1Cre;
R26floxstop-TeNT spinal cord (Figure 1A) and in the Pax6 / spinal
cord (Figure S1 available online). Moreover, it suggests that otheripsilateral inhibitory neurons in the spinal cord secure flexorextensor alternation, either alone or in combination with V1 INs.
Retrograde dextran tracing experiments with fluorescein
isothiocyanate (FITC)-dextran were then used to search for
ipsilaterally projecting inhibitory neurons in the ventral spinal
cord of Pax6 / mice that lack V1 INs (Ericson et al., 1997;
Gosgnach et al., 2006). In E18.5 Pax6 / spinal cords, we
Neuron 82, 138–150, April 2, 2014 ª2014 Elsevier Inc. 139

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Neuronal Control of Flexor-Extensor Alternation

E
A

B

C

D

F

detected ipsilaterally projecting neurons that express the inhibitory vesicular amino acid transporter vGAT (Slc32a1)(Figure 1D). Many of these cells were located rostral to theinjection site (Figure 1E). In view of prior studies showing that
V2b INs are inhibitory (Al-Mosawie et al., 2007; Lundfald
et al., 2007), a Gata3lacZ knockin allele (van Doorninck et al.,1999) was used to test whether the Pax6 / cord contains anormal complement of V2b INs. No difference was detectedin the number of differentiating V2b INs in age-matched E11.5
embryos (Figures 1F–1H; wild-type 52 ± 4 cells versus Pax6 /57 ± 3 cells per section), thereby raising the possibility that V2b
INs secure flexor-extensor alternation in the Pax6 / cord in theabsence of V1 INs.
Characterization of the V2b INs
We then mapped the distribution and number of Gata3+ V2b
INs in spinal cords of GlyT2-GFP (Zeilhofer et al., 2005) andGad1-GFP (Tamamaki et al., 2003) transgenic mice. These
analyses were performed at E13.5, as Gad1-GFP is strongly
downregulated in the ventral horn at later developmental times.
Localization of Gata3 and GlyT2-GFP at E13.5 revealed that
>90% of Gata3+ neurons coexpress GFP (Figures 2A and 2E).
A slightly higher number of Gata3+ V2b INs (95%) expressed
GFP in E13.5 Gad1-GFP reporter mice (Figures 2B and 2E).
Cell counts revealed that Gata3+ V2b INs constitute 19% of
the Gad1-GFP+ cells in the ventral half of the spinal cord
and 9% of the Gad1-GFP+ cells in the whole cord at E13.5(Figure 2F). By comparison V1 INs account for 37% of theventral inhibitory neurons and 17% of the inhibitory neurons in
the whole cord (Figures 2C and 2F).
The medial ventral horn (lamina VIII) was largely devoid of V2band V1 INs (Figures 2A–2D), which is consistent with previous
studies showing lamina VIII is primarily comprised of commissural IN cell types (Harrison et al., 1986; Stepien et al., 2010).
These include Gad1-GFP+/Pax2+ cells (Figure 2D) that are likely
to be V0 INs and dI6 INs (Andersson et al., 2012; Lanuza et al.,2004). Although the majority of ipsilaterally projecting inhibitory
INs in lamina VII (85%) are V1 and V2b INs, these two classes
140 Neuron 82, 138–150, April 2, 2014 ª2014 Elsevier Inc.

Figure 2. Neurotransmitter Phenotype andAxonal Projections of V2b INs(A and B) Gata3 expression in the spinal cords of
GlyT2-GFP (A) and Gad1-GFP (B) transgenic miceat E13.5.
(C) Coexpression of En1 and Gad1.
(D) Coexpression of Pax2 and Gad1.
(E) Quantification of Gata3 and En1 coexpression
with Gad1 and GlyT2. Bars indicate the number ofcells per 30 mm hemicord cross-section (mean ±SEM, n = 3 cords).
(F) Quantification of the proportion of Gad1+ andGlyT2+ neurons that are either V1 or V2b INs at
E13.5.

only account for approximately 30%–35%of the ipsilaterally projecting inhibitory
neurons in the spinal cord as a whole.
The majority of Gad1-GFP+ neurons(56%) in the E13.5 spinal cord arederived from dorsal Lbx1+neurons, which are for the most part,ipsilaterally projecting dI4 and dILA INs (Gross et al., 2002).
To assess the potential contribution that V2b INs make toflexor-extensor motor control, we asked whether motor neurons
innervating the hindlimb musculature receive inhibitory synaptic
inputs from V2b INs. To this end, a Gata3Cre knockin allele was
generated (Figure S2) and crossed with a Cre-conditional Thy1::
floxstop-YFP reporter (Buffelli et al., 2003) to indelibly mark theV2b INs and their axons. V2b INs are primarily located in lamina
VII and have processes that extend throughout the ventral horn
(Figures 3A and 3F). In order to assess the number and distribution of V2b inhibitory synaptic contacts on hindlimb motor
neurons, Cy5-conjugated Cholera Toxin-B (CTB) was injected
into single hindlimb muscles in Gata3Cre; Thy1::floxstop-YFP
mice (Figure 3B). Sections through the lumbar spinal cords ofthese CTB-labeled mice at P15 revealed numerous vGAT+/
GlyT2+ presynaptic contacts on motor neurons within the iliopsoas (IP), gluteus (GL), quadriceps (Q), and posterior bicepssemitendinosus (PB/St) motor pools (Figures 3C and 3E). Motorneurons innervating the gastrocnemius and tibialis anterior
muscles were also decorated with V2b inhibitory contacts(data not shown). V2b inhibitory contacts were found on thesoma of cholinergic V0c INs and on other neurons in lamina VII
and VIII, many of which are likely to be premotor CPG neurons(Figure 3D).
Further analysis of V2b-derived and non-V2b-derived inhibitory contacts onto the soma of CTB-labeled hindlimb motor
neurons revealed that the proportion of V2b-derived vGAT+/
GlyT2+ inhibitory contacts versus all vGAT+/GlyT2+ contacts
varies in a motor-pool-specific manner (Figure 3E), from 23%(IP motor neurons) to 41% (GL motor neurons). V2b INs are
therefore a significant source of the inhibition to hindlimb motor
neurons. A similar analysis of V1 IN-derived contacts (Figure 3E)
revealed that 38%–59% of the inhibitory synaptic contacts on
the soma of hindlimb motor neurons are derived from V1 INs (Figure 3E). Of these, one-third of the contacts can be attributed toRenshaw cell inputs (Figure 3E), which like IaIN synapses are primarily located on the soma and proximal dendrites of motor

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Neuronal Control of Flexor-Extensor Alternation

Figure 3. IaINs Are Derived from V1 andV2b INs

B
A

F

F’
C

F’’

D

G

E

G’

neurons (Jankowska, 1992). Interestingly, the relative proportionof V1 and V2b inhibitory contacts onto hindlimb motor neurons
matches the 2-fold difference in V1 versus V2b cell numbersin the lumbar cord (95 ± 14 V1 cells versus 42 ± 10 V2b cells
per ventral horn section). These analyses point to V1 and V2b
INs being the primary source of inhibition to the somaticcompartment of hindlimb motor neurons. They are also consistent with the idea that the V2b IN population may include IaINs.
Unfortunately, we were unable to determine the number andrelative abundance of V1- and V2b-derived synaptic contacts
on the dendrites of these motor neurons due to the limitationsof the CTB labeling technique.
Our observation that a subpopulation of V2b INs are locatedclose to the border with lamina IX (Figure 3A), where IaINs in
the cat spinal cord are found (Jankowska and Lindström, 1972)
coupled with the persistence of Ia inhibition in mice lacking V1
INs (Wang et al., 2008), prompted us to ask if the V2b INs might
be a source of IaINs. To this end, Gata3Cre; Thy1::floxstop-YFP
reporter mice were used to search for V2b INs that possess
the immunohistochemical features of IaINs. En1Cre; Thy1::floxstop-YFP mice were also used to identify and analyze putative
V1-derived IaINs. Cells were scored as IaINs on the basis ofhigh-density convergent inputs from calbindin+ Renshaw cellaxons and vGluT1+ primary sensory afferents (Alvarez et al.,2005). Approximately 19% of the V2b INs and 29% of the V1
INs in the lumbar spinal cord were categorized as IaINs using
these criteria. These numbers are likely underestimates, assome IaINs may receive vGluT1 inputs on more distal dendrites
that could not be scored in this study. Our findings reveal thatG’’(A) P15 Gata3Cre; Thy1::floxstop-YFP spinal cord
showing V2b INs are located predominantly inlamina VII. Note the V2b cells close to the border oflamina VII/IX.
(B) Schematic showing the protocol for CTB labelingof specific hindlimb motor neuron pools and visualization of inhibitory synaptic contacts.
(C) Section showing the soma and proximal dendrites of two motor neurons (blue) labeled by injecting Cy5-CTB into the PB/St muscles. GlyT2+/
vGAT+ inhibitory synaptic contacts from V2b INs areindicated (arrowheads).
(D) Section showing V2b IN-derived GlyT2+/vGAT+
inhibitory contacts (arrowheads) on the somaof lamina VII neurons. Inset: Inhibitory contacts(arrowheads) from V2b INs on a ChAT+ V0c IN (blue).
(E) Quantification of V1- and V2b-derived synaptic
contacts on iliopsoas (IP), gluteus (GL), posterior
biceps-semitendinous (PB/St), and quadriceps (Q)motor neurons (mean ± SEM, n = 15 cells per motorpool).
(F and G) Sections through the lumbar spinal cord of
P15 Gata3Cre; Thy1::floxstop-YFP (F), and En1Cre;
Thy1::floxstop-YFP (G) mice, showing neurons withthe anatomical features of IaINs. Cells decorated
with calbindin+ (Cb+) (F’ and G’) and vGluT1+ (F’’ andG’’) contacts were scored as IaINs. Cell counts forV2b-derived IaINs and V1-derived IaINs are given
in Table S1.

cells bearing the anatomical characteristics of IaINs develop
from two distinct inhibitory neuron lineages.
Inactivating V2b IN Transmission Compromises FlexorExtensor Coordination
To determine the functional contribution that V2b INs make tomotor coordination and flexor-extensor alternation, we crossed
the Gata3Cre knockin allele (Figure S2) with a conditional TeNT
allele (R26floxstop-TeNT; Zhang et al., 2008), so as to abrogate
inhibitory transmission in all V2b-derived INs. As expected,
wild-type spinal cords produced a strong locomotor-like rhythm
in which L2-iL5 ventral root activity was strictly out of phase(Figure 4A). This alternating L2-iL5 activity is widely considered
to be a proxy measure of flexor-extensor activity (Kiehn, 2006).
Gata3Cre; R26floxstop-TeNT cords also displayed a predominantly
alternating pattern of L2 and iL5 ventral root activity, although
the phasing of this activity was more variable than that seen inwild-type spinal cords (Figure 4B). This suggests that L2-iL5
coordination is mildly degraded in cords lacking V2b-derived
inhibition. Nonetheless, the overall phase relationship between
L2 and iL5 in the Gata3Cre; R26floxstop-TeNT cord remains alternating rather than synchronous. Abrogating V2b transmission
had no effect on left-right alternation between opposing L2
ventral roots (Figure 4B).
We then asked whether the CPG network that generates L2iL5 alternation is fully functional in hemisected spinal cords,
where V2b-derived inhibition is disrupted. Whereas wild-type(Figure 4C) and En1Cre; R26floxstop-TeNT (Figure 1B) hemicords
exhibited an invariant reciprocating pattern of L2-iL5 activity,
Neuron 82, 138–150, April 2, 2014 ª2014 Elsevier Inc. 141

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Neuronal Control of Flexor-Extensor Alternation

Wild-type(whole cord)

A

L2-iL5 (n=15)

Figure 4. Abolishing V2b IN Transmission
Degrades Flexor-Extensor Coordination

L2-cL2 (n=17)L2iL5
r=0.94r=0.90cL2
Gata3Cre; R26floxstop-TeNT(whole cord)

B

L2-iL5 (n=17)

L2-cL2 (n=15)

L2iL5
r=0.73cL2
5s
Wild-type(hemisected)

C

L2-iL5 (n=16)

L2

iL5

r=0.93

Gata3Cre; R26floxstop-TeNT(hemisected)D

r=0.95(A and B) Left: extracellular recordings from
L2, iL5, and cL2 ventral roots of wild-type andGata3Cre; R26floxstop-TeNT from P0 whole cords
following the induction of locomotor activity with
5 mM NMDA and 15 mM 5-HT. Right: circular plots
showing the phase of bursts in iL5 and cL2 withrespect to L2. Each point in the circular plot
represents the average phase for one spinal cord
(25–50 steps). The angular vector (arrow) andr-value (r) for each genotype represents the mean
phase relationship for all recorded cords (n). The
inner circle indicates an r significance value of
p = 0.01. Alternating flexor-extensor (L2-iL5)
activity is observed in wild-type (A) whole cords
and in all but one Gata3Cre; R26floxstop-TeNT (B)whole cord.
(C and D) Left: locomotor-activity following spinal
cord hemisection. Left: the normal pattern of
flexor-extensor (L2-iL5) alternation in wild-type
hemisected cords (C) is markedly reduced in Gata3Cre; R26floxstop-TeNT hemisected cords (D), with
13/20 cords displaying synchronous L2-iL5 activity. Right: circular plots showing the L2-iL5 phasevector points and r-values for the cords in eachexperimental group. n, number of cords analyzed
for each group.

L2-iL5 (n=20)L2iL5
r=0.4320 s

the majority of Gata3Cre; R26floxstop-TeNT hemicords displayed
synchronous L2-iL5 activity (Figure 4D). Thirteen of twenty
Gata3Cre; R26floxstop-TeNT hemicords had mean L2-iL5 vector
points close to 0, which indicates synchronous L2-iL5 activity.
There were, however, some Gata3Cre; R26floxstop-TeNT hemicords(n = 7/20) that retained a loosely alternating pattern of L2-iL5
activity. This suggests that other ipsilateral inhibitory neurons
secure flexor-extensor alternation in the absence of V2b-derived
inhibition. The observation that hemisected Gata3Cre; En1Cre;
R26floxstop-TeNT cords generate a synchronous pattern of L2-iL5
activity (Figure S3) suggests these cells may be V1 INs.
Inactivation of V2b and V1 INs Abrogates Reciprocal
Flexor-Extensor Motor Activity
Our discovery that L2-iL5 coordination is degraded upon abrogating V2b functionality prompted us to test whether V2b and
V1 INs act in a cooperative manner to generate an alternating
flexor-extensor motor rhythm. Gata3Cre; En1Cre; R26floxstop-TeNT
mice induced to locomote with NMDA and 5-HT displayed a
142 Neuron 82, 138–150, April 2, 2014 ª2014 Elsevier Inc.

strictly synchronous pattern of flexorextensor motor activity (Figure 5A; n =14/14 cords). Electrically stimulatingsacral sensory afferents also produced
a synchronous pattern of L2-iL5 locomotor activity in all Gata3Cre; En1Cre;
R26floxstop-TeNT cords tested (Figure 5C;
n = 8/8 cords). By contrast, L2-iL5 activity was always alternating in electricallystimulated wild-type cords (Figure 5B;
n = 7/7 cords). All Gata3Cre; En1Cre;
R26floxstop-TeNT cords displayed a strictly alternating activity
pattern in the contralateral roots (Figures 5A and 5C). Taken
together, these findings show that V1 and V2b INs play anessential role in controlling the phasing of L2-iL5 activity but
do not contribute to left-right alternation.
Altering the Balance of Excitatory and Inhibitory
Transmission Fails to Rescue Alternation in the
Gata3Cre; En1Cre; R26floxstop-TeNT Cord
To rule out the possibility that the synchronous L2-iL5 activity weobserve in the Gata3Cre; En1Cre; R26floxstop-TeNT cord might bedue to a change in the balance between excitation and inhibition
within the locomotor network, we asked if flexor-extensor alternation is rescued when excitatory transmission is decreased or
inhibitory transmission is enhanced in the Gata3Cre; En1Cre;
R26floxstop-TeNT cord. Excitatory transmission was either attenuated by reducing the concentration of NMDA in the perfusate orby adding a low concentration of 6-cyano-7-nitroquinoxaline2,3-dione (CNQX) to the perfusate to weaken non-NMDA

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Neuronal Control of Flexor-Extensor Alternation

A

Gata3Cre; En1Cre; R26floxstop-TeNT

L2-iL5 (n=14)

Figure 5. V1 and V2b INs Function Cooperatively to Establish Flexor-Extensor
Alternation

L2-cL2 (n=13)L2iL5
r=0.90cL2

r=0.9620 s

BWild-typeC

Gata3Cre; En1Cre; R26floxstop-TeNT

L2

L2

iL5

iL5

cL2

cL2

cL5

cL5

L2

L2

cL2

cL2
5s

excitatory transmission, which is the major source of excitatory
drive to the locomotor CPG (Cowley et al., 2005; Whelan et al.,2000). When excitation was decreased by either method, L2iL5 ventral root activity remained synchronous (Figures 6A–6C).
In a second series of experiments, sarcosine was added tothe perfusate to enhance glycinergic transmission. This treatment
also failed to restore alternating L2-iL5 activity in the Gata3Cre;
En1Cre; R26floxstop-TeNT cord (Figure 6D), as did the addition ofnipecotic acid (Figure S4). Left-right alternation was preserved
following all of these manipulations, thus providing further evidence that V1- and V2b-derived inhibition has a selective role insecuring flexor-extensor coordination. By contrast, blocking all
inhibitory transmission in the spinal cord produced an abnormal
pattern of motor activity in which all lumbar ventral roots weresynchronously active (Figure S4). This pattern of activity was
highly irregular rather than rhythmic, suggesting that it does not
represent normal locomotor activity. Taken together, these data
argue that differences in the balance of excitation and inhibition
are unlikely to account for the loss of flexor-extensor alternationin the Gata3Cre; En1Cre; R26floxstop-TeNT cord.
Newborn Mice Lacking V1 and V2b Inhibitory
Transmission Are Unable to Move Their Limbs
To confirm that the loss of alternating L2-iL5 activity seen in vitro
represents an absence of reciprocating flexor-extensor activity(A) Left: example of extracellular recordings from
L2, iL5, and cL2 ventral roots of a P0 Gata3Cre;
En1Cre; R26floxstop-TeNT spinal cord following
application of 5 mM NMDA and 15 mM 5-HT. Right:
phase relationship between L2 and iL5 (L2-iL5)
and between L2 and cL2 (L2-cL2) ventral roots.
The inner circle indicates an r significance value of
p = 0.01.
(B and C) Extracellular recordings from L2, iL5,
and cL2 ventral roots of P0 wild-type (B) and P0
Gata3Cre; En1Cre; R26floxstop-TeNT (C) spinal cords
following sacral dorsal root stimulation. Normal
flexor-extensor (L2-iL5) and left-right (L2-cL2)
alternation is observed in wild-type cords (B).
Flexor-extensor (L2-iL5) alternation is absent in
Gata3Cre; En1Cre; R26floxstop-TeNT cords; however,left-right (L2-cL2) alternation remains intact. n,number of cords analyzed.

in the hindlimb CPG, we examined the
motor activity that is produced in antagonist Q and PB muscles following druginduced locomotion. As inactivating theV1 and/or V2b INs with TeNT results inearly neonatal lethality, these analyses
were performed at P0. Electromyographic(EMG) recordings made from P0 wild-type5s
animals using a limbs-attached spinal
cord preparation (Pearson et al., 2003)
revealed a strictly alternating pattern of
Q-PB activity, as expected (Figure 7A; n = 6/6 mice). By contrast,
Gata3Cre; En1Cre; R26floxstop-TeNT newborn mice displayed rhythmic coactivation of both muscle groups (Figure 7B; n = 7/7mice).
In addition to the loss of flexor-extensor coordinationthat occurs during fictive locomotion, distinct differences werenoted in the motor behaviors of newborn Gata3Cre; En1Cre;
R26floxstop-TeNT mice. Whereas P0 wild-type pups displayed
spontaneous bouts of air stepping in which they extended andflexed their limbs, P0 Gata3Cre; En1Cre; R26floxstop-TeNT pups
displayed no such movements. Instead, all four limbs werecompletely immobilized.
Further behavioral testing of the Gata3Cre; En1Cre;
R26floxstop-TeNT pups revealed clear deficits in a range of motor reflexes, including the righting response that requires a sequence of
flexion and extension movements by all four limbs. Whereas
newborn wild-type pups displayed a strong righting responsewhen placed on their backs (n = 6 mice, three trials each), Gata3Cre;
En1Cre; R26floxstop-TeNT pups were never able to fully rotate from asupine to prone position (n = 6 mice, three trials each). Occasionally,
Gata3Cre; En1Cre; R26floxstop-TeNT pups were able to place both front
feet on the ground, although the time to do so was >18 s for theGata3Cre; En1Cre; R26floxstop-TeNT pups as compared to <5 s forwild-type pups. There was no measureable difference in the righting time for En1Cre; R26floxstop-TeNT and Gata3Cre; R26floxstop-TeNT
pups compared to wild-type pups.
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Neuronal Control of Flexor-Extensor Alternation

A

B

C

D

Figure 6. Loss of Flexor-Extensor Alternation in Gata3Cre; En1Cre;
R26floxstop-TeNT Spinal Cords Is Not due to Changes in the Balanceof Excitation and Inhibition(A) Extracellular recordings from the L2, iL5, and cL2 ventral roots of Gata3Cre;
En1Cre; R26floxstop-TeNT whole-cord preparations at P0 after induction of locomotor activity with 5 mM NMDA and 15 mM 5-HT.
(B and C) Synchronous flexor-extensor (L2-iL5) activity is maintained at lower
NMDA concentrations (B) and after reducing AMPA-dependent excitatorytransmission with 2.5 mM CNQX (C).
(D) Enhancing inhibitory transmission with the glycine uptake inhibitor sarcosine (500 mM) does not rescue L2-iL5 alternation. Note that left-right (L2-cL2)
alternation is preserved under all conditions.

The response of newborn pups to tail pinch was also examined. Newborn mice normally respond to a single tail pinch byflexing and extending their hindlimbs (Figures 7C and 7E). Thissequence of limb movements was observed in wild-type, En1Cre;
R26floxstop-TeNT and Gata3Cre; R26floxstop-TeNT pups (Figure 7E;
n = 43 mice) but never in Gata3Cre; En1Cre; R26floxstop-TeNT
pups (Figures 7D and 7E; n = 10 mice). Gata3Cre; En1Cre;
R26floxstop-TeNT pups did however produce marked lateral
bending movements of the torso (Figure S5), indicating the sensory reflex pathways for sensing noxious stimuli are still intact inthese animals. These behavioral tests, together with the in vitro
spinal cord analyses, demonstrate that mice lose their ability to
execute flexor-extensor-driven limb movements when V1 andV2b inhibitory transmission is abolished.
Abolition of Reciprocal Inhibitory Reflexes in Mice
Lacking V1 and V2b INs
To further understand the nature of the loss of alternating flexorextensor activity in the Gata3Cre; En1Cre; R26floxstop-TeNT mice,
we asked whether these animals retain the inhibitory reflex
pathway between the quadriceps nerve and PB/St motor neurons (Figure 8A; Wang et al., 2008) that is used as a measureof IaIN-mediated reciprocal inhibition in the cat (Hultborn et al.,
1971; Jankowska and Roberts, 1972). In newborn wild-type
144 Neuron 82, 138–150, April 2, 2014 ª2014 Elsevier Inc.

pups (n = 8 mice), inhibitory potentials with synaptic latenciesof 13–17 ms were recorded in 23 of 29 motor neurons, which
we know from our previous analysis are likely to be disynaptic(Wang et al., 2008). All 23 potentials with a disynaptic latency
were hyperpolarizing when the cell was first impaled, demonstrating that these potentials are inhibitory. During the recording,
hyperpolarizing potentials gradually became depolarizing as
the resting potential of the cell became more negative (Figure 8;
see also Wang et al., 2008). To confirm that these potentials are
inhibitory, we performed an additional test on some of these
motor neurons. Short positive current pulses (80–150 pA) were
injected to depolarize the membrane potential by 35–50 mV
and test if the depolarizing currents were converted to
hyperpolarizing potentials. In eight motor neurons from four
mice tested in this manner, positive current injection converted
depolarizing disynaptic potentials to hyperpolarizing potentials(Figure 8B).
This same pathway was examined in P0 Gata3Cre; En1Cre;
R26floxstop-TeNT pups (n = 5 animals), where only 7 of 20 motor
neurons displayed potentials consistent with a disynaptic latency (%16.5 ms). These potentials remained depolarizing, andnone were reversed upon positive current injection (Figures 8C
and 8F; n = 7/7 motor neurons). Thus, while some Gata3Cre; En1Cre; R26floxstop-TeNT mice still possess disynaptic afferent inputs
to motor neurons, the inputs are not inhibitory. Interestingly,
long-latency inhibitory potentials (>18 ms) were observed inmany PB/St motor neurons (see Figures 8D and 8E), demonstrating inhibitory inputs to PB/St motor neurons are not entirely
eliminated when V1 and V2b transmission is abrogated. Finally,
eliminating either V1- or V2b-derived inhibitory transmission
alone failed to abolish inhibitory potentials in PB/St motor neurons that fall within the disynaptic latency window (Figure S6;
Wang et al., 2008). These findings show that the V1 and V2b IN
classes both contribute to reciprocal inhibition in the spinal
cord, and together with the anatomical analysis in Figure 3,
they demonstrate a clear dichotomy in the developmental origin
of IaINs.
DISCUSSION
V1 and V2b INs constitute the core elements of a distributed
inhibitory network that secures the reciprocal pattern of flexorand extensor motor activity that mice use for limb-driven movements. This finding is also consistent with our discovery that
IaINs are derived from V1 and V2b IN progenitors. Our
results suggest that V1 and V2b INs are largely responsible
for this facet of motor coordination, as the inactivation ofother inhibitory neuron populations, including V0 INs andLbx1-derived inhibitory neuron cell types, does not alter
flexor-extensor phasing (Lanuza et al., 2004; Talpalar et al.,2013; M.G., unpublished data). Although flexor-extensor coordination appears to be a primary function of the V1 and V2bINs, we cannot rule out additional roles for V1 and V2b INs inmotor behaviors that cannot be assessed in the isolated spinal
cord preparation.
This study, together with previous studies (Whelan et al., 2000;
Kiehn, 2006), argues that commissural neurons do not contributedirectly to the control of flexor-extensor alternation, as the rodent

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Neuronal Control of Flexor-Extensor Alternation

A Wild-type

B Gata3Cre;En1Cre;R26floxstop-TeNT

E

Figure 7. Defective Limb Movement in
Newborn Gata3Cre; En1Cre; R26floxstop-TeNT
Mice

p <0.001
p <0.01

Q

PB

PB

3

10 s

C

1

2

3

4

Number of flexionextension movements

Q

ns

2

1

*

0
Wild-type (n=18)

D

1

2

3

4

En1Cre; R26floxstop-TeNT (n=12)
Gata3Cre; R26floxstop-TeNT (n=13)* Gata3Cre; En1Cre; R26floxstop-TeNT (n=10)

spinal CPG still produces a reciprocating pattern of L2-iL5 flexorextensor activity when all commissural connections are severed.
Interestingly, the degradation of flexor-extensor alternation ismore pronounced in the hemisected Gata3Cre; R26floxstop-TeNT
cord compared to the intact Gata3Cre; R26floxstop-TeNT cord.
This raises the possibility that some commissural INs provide
excitatory drive to V1 INs. V1-derived IaINs are contacted by glutamatergic V3 INs (Zhang et al., 2008), raising the possibility thatV3 commissural INs subserve this function.
Contribution of V1 and V2b IN Inputs to Motor Neuron
Inhibition
The high density of V1 and V2b inhibitory contacts on the soma
and proximal dendrites of motor neurons suggests that V1 andV2b INs provide most of the inhibition to the soma of these cells(Figure 3). They are therefore likely to strongly gate excitatory inputs to motor neurons. Other studies show inhibitory commissural INs (Kiehn, 2006; Lanuza et al., 2004) and inhibitory INs
in the dorsal horn (Jankowska, 1992; Tripodi et al., 2011; Wilson
et al., 2010) also synapse with motor neurons. The location ofthese synapses is not known, and a significant fraction of these
may be on motor neuron dendrites (see Ornung et al., 1998; Shigenaga et al., 2005). Given our current inability to score inhibitory contacts on dendrites, the relative contribution thatcommissural INs, dorsal inhibitory INs, and V1/V2b INs maketo motor neuron inhibition remains to be determined. Nonetheless, our demonstration that V1 and V2b INs are strictly required
for flexor-extensor alternation (see Figures 5 and 7), coupledwith the abundance of V1 and V2b IN inputs on motor neuron
soma, suggests that the actions of these other inhibitory cell
types on flexion-extension movements are likely to be highly
dependent on V1 and V2b INs. It should also be noted that V1
and V2b INs provide inhibitory inputs to other CPG neurons(Figure 3), including excitatory neurons that are also likely toplay an important role in shaping flexor-extensor activity(Figure 9).

(A and B) EMG recordings from knee extensor(quadriceps, Q) and knee flexor (posterior biceps,
PB) muscles of newborn P0 mice following induction of locomotor activity in a limbs-attached spinal
cord preparation with a cocktail of 10 mM NMDA,
25 mM 5-HT, and 25 mM dopamine. Quadriceps (Q)and posterior biceps (PB) muscle activity is alternating in wild-type animals (A) and synchronous in
Gata3Cre; En1Cre; R26floxstop-TeNT mice (B).
(C) A series of time-lapse images of P0 wild-type
pups showing flexion-extension movements of thehindlimbs in response to tail pinch.
(D) Gata3Cre; En1Cre; R26floxstop-TeNT mutant animals show no flexion-extension movements, andtheir limbs are immobile.
(E) Number of flexion-extension movements in
response to a single tail pinch in wild-type pups
and age-matched En1Cre; R26floxstop-TeNT, Gata3Cre;
R26floxstop-TeNT and Gata3Cre; En1Cre; R26floxstop-TeNT
animals. Student’s t test. Error bars, SEM.

Role of Inhibition in Establishing Flexor-Extensor
Alternation
Prior studies demonstrating flexor-extensor alternation is only
abrogated upon blocking all inhibitory transmission in the cordhave led to speculation that the relative level of inhibition versus
excitation in the spinal motor network may be the mechanismthat generates an alternating flexor-extensor motor output
from the walking CPG. Our data argue against this possibility,
as pharmacological manipulations that reduce excitation in
the Gata3Cre; En1Cre; R26floxstop-TeNT cord spinal cord, whilechanging the amplitude and frequency of the motor rhythm, do
not alter the phasing of L2-iL5 flexor-extensor-related activity(Figure 6). Furthermore, the motor pattern that emerges whenbicuculline and strychnine are used to block inhibitory transmission differs in two key aspects from the pattern observed
following V1 and V2b IN inactivation. First, widespread pharmacological blockade of glycinergic and GABAergic neurotransmission results in irregular episodes of high-amplitude motor
neuron bursting interspersed with periods of little of no activity(Figure S4). The periodicity of these bursts is highly variable,arguing that this activity is not rhythmic. Second, in contrast to
the Gata3Cre; En1Cre; R26floxstop-TeNT cord, where left-right alternation is preserved (Figures 4 and 5), abrogating inhibition
causes all the lumbar ventral roots to burst synchronously(Figure S4; see also Cowley and Schmidt, 1995; Kremer andLev-Tov, 1997).
Functional Organization of the Walking CPG
This study demonstrates two important principles about thecellular organization of the walking CPG. First, flexor-extensor
activity is not controlled by a single class of neurons but isinstead encoded by the composite actions of V1 and V2b INs.
This suggests that the evolutionary transition from a swimming
circuit to a walking circuit was not accomplished by simply
adding a new cell type with a dedicated role in flexor-extensor
control. Second, it shows V1 and V2b INs are not part of the
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Neuronal Control of Flexor-Extensor Alternation

Figure 8. Loss of Reciprocal Inhibitory Reflexes in Mice Lacking V1 and V2b INs(A) Schematic showing disynaptic pathway from
quadriceps (Q) afferent nerve to posterior bicepssemitendinosus (PB/St) motor neurons.
(B–E) Intracellular recordings of synaptic responses in PB/St motor neurons were elicited bystimulating the Q nerve (3–53 threshold). Hyperpolarizing inhibitory potentials often become depolarizing as the resting potential (indicated at the
beginning of each trace) increased. An inhibitory
potential (IPSP) in a wild-type mouse that is disynaptic (13–17 ms) is shown in (B). In Gata3Cre;
En1Cre; R26floxstop-TeNT mice, synaptic potentials
with disynaptic latencies (13–17 ms) either
were depolarizing and could not be reversed withcurrent injection (C) or were long latency (D and E).
A hyperpolarizing response (23 ms) that is inconsistent with disynaptic inhibition is shown in (E).
(F and G) Frequency of disynaptic inhibitory potentials in control and Gata3Cre; En1Cre; R26floxstopTeNTpups before (F) and after (G) current injection.
The significance between the number of disynaptic IPSPs in wild-type versus Gata3Cre;
En1Cre; R26floxstop-TeNT samples was determinedusing a Mann-Whitney two-tailed test. The sample
size is indicated below each graph.

rhythm-generating network and rhythm generation does not
require an alternating pattern of flexor-extensor activity. Ourdemonstration that IaINs and Renshaw cells are dispensable
for rhythm generation also argues against half-center models
that position IaINs and Renshaw cells at the center of arhythm-generating network (Miller and Scott, 1977; Pratt andJordan, 1987; Talpalar et al., 2011). Such models fail to accountfor the persistence of the motor rhythm when flexor-extensor
alternation is disrupted (Figures 4 and 5), and they are difficult
to reconcile with the more complex patterns of motor activity
and resetting-deletions that occur during in vivo locomotion(McCrea and Rybak, 2008).
The pattern of locomotor activity produced by the Gata3Cre;
En1Cre; R26floxstop-TeNT cord is consistent with a two-level
CPG model that contains separate rhythm-generating leveland pattern formation layers (McCrea and Rybak, 2008). In
this configuration, V1 and V2b INs would be embedded in thepattern formation layer, where they would be a source of phasic
inhibition to flexor and extensor motor neurons. They would
also provide inhibitory feedback to the rhythm-generating
layer to produce an alternating pattern of excitation to flexorand extensor motor neurons (Figure 9). Such a model is consistent with our demonstration that the flexor-extensor rhythm
collapses into a single synchronous rhythm when V1- andV2b-derived inhibition is abrogated. This also leads us to
speculate that the synchronous rhythmic activity seen in the
Gata3Cre; En1Cre; R26floxstop-TeNT cord might be an atavistic
rhythm, which is equivalent to a primitive swimming rhythm
that lacks a differential flexor-extensor component. Froman evolutionary perspective, this would provide a far more
146 Neuron 82, 138–150, April 2, 2014 ª2014 Elsevier Inc.

satisfactory solution to the issue of rhythm generation in limbed
vertebrates than those presented by flexor-extensor half-centermodels.
Role of Ia Inhibitory INs in Flexor-Extensor Coordination
Our study leaves open the question as to whether flexorextensor alternation is broadly encoded by the V1 and V2b populations as a whole or by the activity of specific V1 and V2b cell
types, such as IaINs. The V1 and V2b IN classes are eachcomposed of multiple cell types (Goulding, 2009). These include
putative IaINs that constitute 29% and 19% of lumbar V1 andV2b cells, respectively. Renshaw cells make up a further 10%–
12% of the V1 population (Sapir et al., 2004). The V1 and V2b
IN classes are also likely to include other inhibitory subtypes
with different connections and function. Consequently, caution
needs to be exercised when ascribing the role of flexor-extensor
coordination to a particular cell type, such as IaINs. Although
IaINs possess the necessary attributes to play a prominent rolein flexor-extensor control (Jankowska, 1992; Feldman and
Orlovsky, 1975), their exact contribution to flexor-extensor alternation has yet to be determined. Our discovery that cells possessing the anatomical and functional features of IaINs arise
from V1 and V2b INs (Figures 3 and 8) is consistent with a rolefor IaINs in flexor-extensor alternation. However, we currently
lack the means to genetically isolate IaINs and evaluate their
exact contribution to flexor-extensor motor behaviors. Furthermore, it is likely that flexor-extensor control, rather than being
restricted to a dedicated physiological cell type, is instead
distributed over multiple V1 and V2b cell types. In this respect,
it is worth noting that fish with a primitive appendicular system

Neuron
Neuronal Control of Flexor-Extensor Alternation

In summary, this study provides evidence that the flexorextensor system is organized around inhibitory V1 and V2b IN
cell types, including IaINs, thereby demonstrating a developmental dichotomy in the composition of the spinal inhibitory
network that secures flexor-extensor alternation in walking vertebrates. We would like to propose that the distribution of
flexor-extensor control between V1 and V2b IN cell types arose
as a result of the evolutionary reorganization of the swimming
CPG circuit, and it was necessary for the emergence of movablebilateral appendages.
EXPERIMENTAL PROCEDURES
Animals
The generation, characterization, and genotyping of the En1Cre, Gata3lacZ,
Pax6 / , R26floxstop-lacZ, R26floxstop-TeNT, R26floxstop-GFP, R26floxstop-Td-Tomato,Gad1-GFP, GlyT2-GFP, and Thy1::floxstop-YFP mice have been described
previously (Buffelli et al., 2003; Gosgnach et al., 2006; Madisen et al., 2010;
Stam et al., 2012; Tamamaki et al., 2003; van Doorninck et al., 1999; Zeilhofer
et al., 2005; Zhang et al., 2008). Animal studies were approved by the Salk
Animal Care and Use Committee and performed in accordance with theguidelines stipulated by the National Institutes of Health.

Figure 9. Model for V1 and V2b IN Control of Flexor-Extensor Motor
Activity
Prospective model for how the limb CPG is functionally organized with respect
to V1 and V2b INs. Arrows indicate excitatory pathways. Lines with filled
spheres indicate inhibitory pathways. Weak excitatory connections between
presumptive rhythm-generating excitatory neurons result in the synchronous
rhythmic excitation of flexor and extensor motor neurons when V1 and V2binhibition is absent.

do not possess specialized muscle spindles (Young, 1981),
which is consistent with the idea that IaIN specialization
occurred after the initial recruitment of V1 and V2b INs for
flexor-extensor control.

Relationship of V1 and V2b INs to Spinal Neurons
in Swimming Vertebrates
Our results reveal that the neurons mice use to secure alternating
flexor-extensor driven limb movements share a common developmental origin with neurons that are part of the motor circuitry
aquatic vertebrates use for swimming. The developmental
homologs of V1 and V2b INs are present in the spinal cords ofswimming vertebrates, with V2b INs being molecularly homologous to VeLD neurons (Batista et al., 2008; Peng et al., 2007)
and V1 INs having as their homologs, aIN cells in Xenopus andCiA cells in zebrafish (Higashijima et al., 2004; Li et al., 2004).
aIN and CiA inhibitory neurons are rhythmically active duringswimming (Higashijima et al., 2004; Li et al., 2004), and they
are candidates for the longitudinal inhibitory pathways that
regulate intersegmental delay and the swim cycle period (Gabriel
et al., 2008; Tegnér et al., 1993; Tunstall and Roberts, 1994). This
leads us to posit that V1 and V2b INs were co-opted to produce
an alternating motor output from motor neurons that innervate
the fin adductor/abductor muscles in primitive fish.

Generation of Gata3Cre MiceSequences encoding for Cre recombinase and FRT-PGKneopA-FRT were inserted into the ATG of exon 2 of the Gata3 gene by recombineering. 2A ES cells
were electroporated with a linearized targeting vector and screened for homologous recombination. Two recombinant knockin cell lines gave germline
transmission and the expected pattern of Cre recombination (see Figure S2).
In Situ Hybridization and Immunohistochemistry
X-gal staining, immunohistochemistry, and digoxigenin in situ hybridization
analyses were performed using standard techniques (see Lanuza et al.,2004). In situ images were collected with a Zeiss Axioskop microscope using
brightfield illumination. Fluorescence antibody stainings of spinal cord sections were imaged with Zeiss LSM510 and Olympus Fluoview 1000 confocal
microscopes. All images were assembled using Photoshop software.
Retrograde Labeling of Neurons
Fluorescent Dextran Labeling
Fluorescent dextran labeling was performed as described previously (Sapir
et al., 2004). Further details can be found online in the Supplemental Experimental Procedures.
Cholera Toxin-B Labeling of Motor Neurons
Individual muscles were injected unilaterally with Cy5-Cholera Toxin-B (CTB)
as previously described by Shneider et al. (2009). Antibodies to the presynaptic markers vGAT and GlyT2 were used in combination with V1 and V2bIN-specific YFP reporters to visualize putative inhibitory synaptic contacts
on Cy5-CTB-labeled motor neurons. Calbindin (Cb) immunohistochemistry
was used to score Renshaw cell-derived contacts on motor neurons in thesame pool.
Electrophysiology
Electroneurogram Recordings
Electrophysiological recordings were performed on embryonic E18.5 or postnatal P0 mice. Animals were anesthetized, decapitated, and spinal cords weredissected out in ice-cold Ringers solution. Ventral root electroneurogram(ENG) recordings were made as described previously (Lanuza et al., 2004).
Rhythmic locomotor-like activity was induced either by adding NMDA (5 mM)
and 5-hydroxytryptamine (5-HT; 15 mM) to the Ringer’s perfusate or by electrical stimulation. For the latter, a train of 40 individual pulses (40–50 mA in
amplitude and 500 ms in duration) were applied to the S1-S3 dorsal root on
one side of the cord at 500 ms intervals. Signals were amplified, band-passfiltered, digitized, and collected with Axoscope and pCLAMP software.

Neuron 82, 138–150, April 2, 2014 ª2014 Elsevier Inc. 147

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Neuronal Control of Flexor-Extensor Alternation

Analysis of Locomotor Activity
Step cycle phase values (mean of 25–50 locomotor cycles) were calculated as
described previously (Lanuza et al., 2004). Circular statistics (Zar, 1974) wereused to determine the phasing and coupling strength between the L2, iL5,
and cL2 ventral roots. Vector points (black dots) representing the mean phasevalues for 25–50 steps were determined for each cord. Angular vectors (bluearrows) illustrating the mean phase and concentration of phase values aroundthe mean were then calculated for each experimental group.
Electromyogram Recordings
Electromyogram (EMG) recordings were performed on P0 mice. P0 pups were
decapitated and dissected in ice-cold Ringer’s solution. The cervical andthoracic spinal cord was exposed from the spinal column, while the trunk
below diaphragm and hind legs being was kept intact. For recordings, twobipolar tungsten electrodes were inserted separately into thigh (Q) and posterior hamstring (PB/St) muscles. Locomotor activity was induced in the hindlimbs-attached preparation by adding NMDA (10 mM), 5-HT (25 mM), anddopamine (50 mM) to the Ringer’s perfusate (Pearson et al., 2003). EMG signals
were collected and filtered as described above for ventral root ENG
recordings.
Intracellular Recordings of Muscle Afferent Input to Motor Neurons
The preparation used to assess the disynaptic inhibitory inputs from muscle
afferents to motor neurons in neonate mice is shown schematically in Figure 8A. The methods used for recording and analyzing synaptic potentials
are described in Wang et al. (2008). Positive current pulses (80–150 pA)
were injected into some cells to depolarize them by 35–50 mV and test whether
they are inhibitory, as assayed by the conversion of depolarizing synaptic
potentials into hyperpolarizing potentials.
Tail Pinch Test
Behavioral tests were performed on awake P0 pups. Newborn pups wereplaced back down on a flat surface and the tail gently pinched by a pair offine forceps. Each episode was video recorded, and the number of limb/torso
movements for each episode was counted. Three replicates were performed
for each pup.
Data Analysis and Statistical Tests
Circular statistics were used to evaluate the uniformity, quality, and couplingstrength of ventral rooting activities as previously described (Lanuza et al.,
2004; Zar, 1974). The angular value and step duration for each spinal cord
was calculated for 25–50 cycles of locomotor activity. Rayleigh’s test was
performed to examine the uniformity within each group of spinal cord recordings. The difference of angular value between each group was examined by
the Watson-William’s test. Student’s t test (with Bonferroni correction whenneeded), ANOVA, and Mann-Whitney tests were used for all other statisticalanalyses. p values less than 0.01 were considered highly significant.

their thoughtful comments and criticisms. This research was supported
by grants from the National Institutes of Health (R37-NS037075, P01NS031249, R01-NS080586, and R01-NS047357), the Human Frontier Science
Program, and the Christopher and Dana Reeve Foundation. G.M.L. was
supported by an HFSP postdoctoral fellowship.
Accepted: January 24, 2014
Published: April 2, 2014
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SUPPLEMENTAL INFORMATION

Cowley, K.C., Zaporozhets, E., Maclean, J.N., and Schmidt, B.J. (2005). Is
NMDA receptor activation essential for the production of locomotor-like activity in the neonatal rat spinal cord? J. Neurophysiol. 94, 3805–3814.

Supplemental Information includes Supplemental Experimental Procedures,
six figures, and one table and can be found with this article online at http://dx.doi.org/10.1016/j.neuron.2014.02.013.

Crone, S.A., Quinlan, K.A., Zagoraiou, L., Droho, S., Restrepo, C.E., Lundfald,L., Endo, T., Setlak, J., Jessell, T.M., Kiehn, O., and Sharma, K. (2008). Genetic
ablation of V2a ipsilateral interneurons disrupts left-right locomotor coordination in mammalian spinal cord. Neuron 60, 70–83.

AUTHOR CONTRIBUTIONS

Eccles, J.C., Fatt, P., and Landgren, S. (1956). Central pathway for direct
inhibitory action of impulses in largest afferent nerve fibres to muscle.
J. Neurophysiol. 19, 75–98.

Jingming Zhang undertook all of the electrophysiological analyses. The recordings in Figure 8 were performed by Zhi Wang, with help from Jingming
Zhang. Guillermo Lanuza generated and characterized the Gata3Cre knockinmice used in this study. He also performed the anatomical and neurotracing
analyses with help from Valerie Seimbab. Both co-first authors helped withthe experimental design and writing of the manuscript.

Ericson, J., Rashbass, P., Schedl, A., Brenner-Morton, S., Kawakami, A., vanHeyningen, V., Jessell, T.M., and Briscoe, J. (1997). Pax6 controls progenitor
cell identity and neuronal fate in response to graded Shh signaling. Cell 90,
169–180.

ACKNOWLEDGMENTS

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We would like to thank Simon Gosgnach for his early contribution to theanalysis of the Pax6 mutant mice. We also thank Tom Jessell, Chris Kintner,
Greg Lemke, John Thomas, Lidia Garcia-Campmany, and James Flynn for

148 Neuron 82, 138–150, April 2, 2014 ª2014 Elsevier Inc.

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