Neuronal activity in the isolated mouse spinal cord during ...pages.nbb.cornell.edu/neurobio/harris-warrick/143.pdf · The Journal of Physiology ... low-calcium Ringer solution composedof111mMNaCl,3.08mMKCl,25mM

J Physiol 590.19 (2012) pp 4735–4759 4735

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Neuroscience Neuronal activity in the isolated mouse spinal cord during

spontaneous deletions in fictive locomotion: insights intolocomotor central pattern generator organization

Guisheng Zhong1, Natalia A. Shevtsova2, Ilya A. Rybak2 and Ronald M. Harris-Warrick1

1Department of Neurobiology and Behavior, Cornell University, Ithaca, NY, USA2Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, PA, USA

Key points

• The organization of the spinal circuitry responsible for the generation of locomotor rhythmand control of locomotion in mammals is largely unknown, though several types of spinalinterneurons involved in the rodent locomotor network have been identified.

• Ventral root recordings of spinal motoneurons during fictive locomotion in the isolated mousespinal cord show spontaneous deletions of activity. The majority of deletions in the isolatedneonatal mouse spinal cord are non-resetting: they do not change the phase of subsequentmotor cycles. Flexor and extensor motoneurons express asymmetric responses during deletions:flexor deletions are accompanied by tonic ipsilateral extensor activity, while extensor deletionsdo not perturb rhythmic ipsilateral flexor activity. Non-resetting deletions on one side of thecord do not perturb rhythmic activity on the other side of the cord and can occur in isolatedhemicords.

• We have characterized the activity of motoneurons and identified interneurons duringspontaneous motor deletions. The motoneurons and a subset of V2a interneurons fallsilent during non-resetting motor deletions while a second subset of V2a interneurons andcommissural interneurons continue unperturbed rhythmic firing. This allowed us to suggesttheir involvement at different levels of the locomotor network operation.

• We have developed a computational model of the central pattern generator that reproduces,and proposes a mechanistic explanation for, our experimental results. The model providesnovel insights into the organization of spinal locomotor networks.

Abstract We explored the organization of the spinal central pattern generator (CPG) forlocomotion by analysing the activity of spinal interneurons and motoneurons during spontaneousdeletions occurring during fictive locomotion in the isolated neonatal mouse spinal cord,following earlier work on locomotor deletions in the cat. In the isolated mouse spinal cord,most spontaneous deletions were non-resetting, with rhythmic activity resuming after an integernumber of cycles. Flexor and extensor deletions showed marked asymmetry: flexor deletions wereaccompanied by sustained ipsilateral extensor activity, whereas rhythmic flexor bursting was notperturbed during extensor deletions. Rhythmic activity on one side of the cord was not perturbedduring non-resetting spontaneous deletions on the other side, and these deletions could occurwith no input from the other side of the cord. These results suggest that the locomotor CPGhas a two-level organization with rhythm-generating (RG) and pattern-forming (PF) networks,in which only the flexor RG network is intrinsically rhythmic. To further explore the neuro-nal organization of the CPG, we monitored activity of motoneurons and selected identified

C© 2012 The Authors. The Journal of Physiology C© 2012 The Physiological Society DOI: 10.1113/jphysiol.2012.240895

4736 G. Zhong and others J Physiol 590.19

interneurons during spontaneous non-resetting deletions. Motoneurons lost rhythmic synapticdrive during ipsilateral deletions. Flexor-related commissural interneurons continued to firerhythmically during non-resetting ipsilateral flexor deletions. Deletion analysis revealed twoclasses of rhythmic V2a interneurons. Type I V2a interneurons retained rhythmic synaptic driveand firing during ipsilateral motor deletions, while type II V2a interneurons lost rhythmic synapticinput and fell silent during deletions. This suggests that the type I neurons are components of theRG, whereas the type II neurons are components of the PF network. We propose a computationalmodel of the spinal locomotor CPG that reproduces our experimental results. The results mayprovide novel insights into the organization of spinal locomotor networks.

(Resubmitted 13 July 2012; accepted 5 August 2012; first published online 6 August 2012)Corresponding author R. M. Harris-Warrick: Department of Neurobiology and Behavior, Cornell University, W 159Seeley G. Mudd Hall, Ithaca, NY 14853, USA. Email: [email protected]

Abbreviations BES, brain electrical stimulation; CFP, cyan fluorescent protein; CIN, commissural interneuron;CPG, central pattern generator; MLR, midbrain locomotor region; MN, motoneuron; PF, pattern-forming; RG,rhythm-generating.

Introduction

The spinal cord of vertebrates contains special neuralnetworks, called central pattern generators (CPGs), whichcan organize rhythmic locomotor activity in the absenceof supraspinal and sensory inputs (Graham-Brown, 1911;Grillner, 1981, 2006; Kiehn, 2006; Jankowska, 2008;Goulding, 2009). Recently developed genetic methodshave been used to identify and characterize multiple inter-neuron types that are involved in CPG operation (Lanuzaet al. 2004; Butt et al. 2005; Hinckley et al. 2005; Wilsonet al. 2005; Gosgnach et al. 2006; Crone et al. 2008, 2009;Zhang et al. 2008; Kwan et al. 2009; Zagoraiou et al. 2009;Dougherty & Kiehn, 2010; Zhong et al. 2010). However,the overall organization of the locomotor CPG and thepattern of synaptic interconnections between the key inter-neuron types remain largely unknown.

One way to study the organization of a neural network isto analyse the consequences of perturbations that disturbits output. In the case of the locomotor CPG, whichgenerates a rhythmic locomotor pattern with alternatingactivity of antagonist motoneuron groups, one class ofsuch perturbations is motor ‘deletions’. Deletions arespontaneous errors in the rhythmic locomotor patternwhen a set of synergist motoneurons (for example flexormotoneurons on one side) loses rhythmic firing or fallssilent during a time period when they are normally active.Deletions were observed during cat locomotion (Duysens,1977; Grillner & Zangger, 1979) and were analysed indetail during turtle scratch (Stein & Daniels-McQueen,2002, 2004; for review see Stein, 2008). Lafreniere-Roula& McCrea (2005) recently studied deletions during fictivelocomotion and scratch in the decerebrate cat. Theyfound that deletions in activity of one set of synergistmotoneurons (for example, hindlimb extensors) wereusually accompanied by either sustained activity orcontinuing bursting in antagonist ipsilateral motoneurons

(for example hindlimb flexors). Interestingly, during themajority of these deletions, the phase of the rhythm afterthe deletion did not change, i.e. the rhythmic activityresumed after an integer number of missed cycles. Thesedeletions were called non-resetting deletions. A minorityof deletions (called resetting) did show rhythm resettingwhich was recognized by a shift in the phase of the motorbursts after the deletion.

Rybak and McCrea proposed a computational modelof the mammalian locomotor CPG that could reproducethese results and explain the origin of resetting andnon-resetting deletions (Rybak et al. 2006a,b; McCrea &Rybak, 2007, 2008). In this model, the locomotor CPGhas two functional levels: a half-centre rhythm generator(RG), performing a ‘clock’ function and determining therhythmic output of the system (oscillation frequency,phase durations), and pattern formation (PF) networks,which receive rhythmic drive from the RG and coordinatethe phasing and intensity of activity of the differentmotoneuron populations. The concept of a two-level CPGorganization containing separate RG and PF networks hadbeen suggested by a number of previous studies (Perret& Cabelguen, 1980; Kriellaars et al. 1994, reviewed byMcCrea & Rybak, 2008; Guertin, 2009) and experimentallysupported by other studies (Guertin et al. 1995; Perreaultet al. 1995; Lafreniere-Roula & McCrea, 2005, reviewedby Guertin, 2009). According to the Rybak-McCreamodel, failure of activity at the RG level should generallyresult in a resetting deletion, while failure of activity atthe PF level should result in a non-resetting deletion.Therefore, this two-level CPG model could reproduce allthe resetting and non-resetting deletions observed duringfictive locomotion in decerebrate adult cats (Rybak et al.2006a; McCrea & Rybak, 2007).

In this study, we analysed spontaneous deletionsoccurring during fictive locomotion in the isolatedneonatal mouse spinal cord. Unlike the previous work

C© 2012 The Authors. The Journal of Physiology C© 2012 The Physiological Society

J Physiol 590.19 Neuronal activity during locomotor deletions 4737

in the decerebrate cat, we monitored flexor and extensormotoneuron activity on the ventral roots from both leftand right sides of the spinal cord. We found that thereis a marked asymmetry in the pattern of antagonistmotoneuron activity during non-resetting flexor deletionsas opposed to extensor deletions in the neonatal mousespinal cord. We also found that non-resetting spontaneousdeletions on one side of the cord were almost neveraccompanied by disturbances in rhythmic motor activityon the other side of the cord. Simultaneously, we were ableto record the activity of several classes of identified spinalinterneurons and motoneurons, and investigate theirbehaviour during non-resetting flexor deletions observedduring evoked fictive locomotion. Based on the analysisof these deletions we propose a computational modelthat can reproduce, and suggest explanations for, ourexperimental results. Our study may provide insights intothe organization of the locomotor CPG in the neonatalmouse spinal cord.

Methods

Spinal cord preparations with and without attachedbrainstem

Experiments were performed using the isolated spinalcord, with or without brainstem attached, from neonatal(postnatal day (P)P0–P3) wild-type ICR strain orChx10::eCFP mice, which were generated from the ICRstrain, and the Chx10 interneurons in the spinal cord aregenetically labelled with cyan fluorescencee proteins (CFP)(Zhong et al. 2010). The animal protocol was approvedby the Institutional Animal Care and Use Committee atCornell University and was in accordance with NationalInstitutes of Health guidelines. The spinal cord dissectionwithout brainstem attached was performed as describedpreviously (Zhong et al. 2006a,b, 2007). Briefly, neonatalmice were decapitated and eviscerated in cold oxygenated(95% O2 and 5% CO2) low-calcium Ringer solutioncomposed of 111 mM NaCl, 3.08 mM KCl, 25 mMNaHCO3, 1.18 mM KH2PO4, 3.5 mM MgSO4, 0.25 mMCaCl2 and 11 mM glucose. The spinal cord, extendingfrom T8/9 to S3/4, was removed and pinned dorsal-side-upin a recording chamber superfused with oxygenated Ringersolution, composed of 111 mM NaCl, 3.08 mM KCl,25 mM NaHCO3, 1.18 mM KH2PO4, 1.25 mM MgSO4,2.52 mM CaCl2 and 11 mM glucose. To visualize theV2a interneurons in the spinal cord, we removed asmall dorsal part of the L2 segment with fine scissorsduring the dissection. In the brainstem–spinal cord pre-paration, after evisceration, a piece of the skull wasremoved and a rostral cut was made between themesencephalon and the diencephalon; the brainstem andattached spinal cord were carefully isolated, bilaterallyintact (Zhong et al. 2011). The preparation was pinned

dorsal-side-up in the lumbar spinal cord region, whilethe brainstem was positioned ventral-side-up by twistingthe thoracic/cervical segments by 180 deg. With thesemanipulations, we were able to record the membraneproperties of V2a interneurons from the cut dorsal surfacein the lumbar region while electrically stimulating theventral brainstem area to trigger the fictive locomotion.All experiments were performed at room temperature(20–23◦C).

Fictive locomotion in isolated spinal cord

We recorded extracellular motoneuron activity withfire-polished glass suction electrodes on the ventralroots. Fictive locomotion, characterized by the alternationof left/right and ipsilateral flexor (L2)/extensor (L5)ventral root activity, was triggered in two differentways. First, NMDA (5–10 μM) and 5-HT (5–10 μM)were superfused over the recording chamber, reliablyinducing fictive locomotion in the isolated spinal cord.Second, fictive locomotor activity was induced by tonicelectrical stimulation of the brainstem. An ACSF-filledglass electrode, with a tip diameter of∼100 μm, was placedon the ventral surface of the brainstem, and tonic brainelectrical stimulation (BES) was applied (2–4 Hz, 4–5 ms,1–5 mA). Rhythmic locomotor activity was triggered after3–8 s of stimulation. In this series of experiments, onerecording suction electrode was placed on the ipsilateralL2 (iL2) ventral root to monitor BES-induced fictivelocomotion. Rhythmic burst activity from the ventralroot was bandpass-filtered (100 Hz to 1 kHz). The fictivelocomotion from neonatal Chx10::eCFP spinal cord iscomparable to that wild-type (Zhong et al. 2010).

Electrophysiological recording from spinalinterneurons and motoneurons

Commissural interneurons (CINs) and motoneurons(MNs) were recorded from isolated wild-type neonatalmouse spinal cords with blind patch clamp recordings. Toidentify the CIN type, we used two suction electrodes tostimulate the split rostral and caudal hemicords on theopposite side from the intracellular recording. Collisiontests were employed to identify whether the recordedneurons were ascending or descending CINs (Zhong et al.2006a,b, 2007). In the current study, only commissuralinterneurons which showed highly rhythmic activityduring NMDA- and 5-HT-induced fictive locomotionwere included for analysis. To identify flexor motoneurons,we used a suction electrode to stimulate the ipsilateral L2ventral root in the same segment as the neuronal recording,and verified the motoneurons with a collision test oforthodromic action potentials evoked by the recordingelectrode in the soma and antidromic action potentials



evoked by stimulation of the ipsilateral L2 ventralroot, as described previously (Zhong et al. 2007). V2ainterneurons were recorded from the L2 segment ofneonatal spinal cords with partial dorsal horn removalfrom Chx10::eCFP mice during fictive locomotion,as described above. Whole-cell recordings were madewith electrodes pulled from thick-walled borosilicateglass (WPI, Sarasota, FL, USA) on a vertical puller(Narishige, Japan) with resistances of 6–10 M�; thesewere lowered through the cut dorsal surface of the L2segment to V2a interneurons identified by their CFPfluorescence. The pipette solution contained 138 mM

potassium gluconate, 10 mM Hepes, 5 mM ATP-Mg,0.3 mM GTP-Li and 0.0001 mM CaCl2 (pH adjustedto 7.4 with KOH). The seal resistance obtained beforerecordings was always greater than 2 G�. Patch clamprecordings were made with a Multiclamp 700B amplifier(Molecular Devices, Sunnyvale, CA, USA) and weredriven by Clampex (pCLAMP 9, Molecular Devices).Data were filtered at 20 kHz. Intracellular recordings wereaccepted for analysis if the action potential overshootwas >0 mV.

Data analysis

Clampfit 9.0 (Molecular Devices), Microsoft Excel andSpike2 (Cambridge Electronic Design, Cambridge, UK)were used for data analysis. A cycle of motor nerve activitystarted with the onset of an iL2 ventral root burst andended at the onset of the next iL2 burst; these onsets weredetermined by a custom-made program in Spike2 to detectwhen the rectified signal exceeded the average noise levelbetween bursts by a preset amount. Data were analysedin preparations that showed stable rhythmic ventralroot activity. Individual spinal neurons were recordedfor more than 20 min during NMDA/5-HT-inducedlocomotor-like activity and for more than 5 min duringtonic BES-induced fictive locomotion. The extracellularventral root recordings were rectified and smoothedwith a time constant of 0.2 s, and analysed usingcustom-written programs in Spike2. The relationshipbetween ventral root activity and neuronal membranepotential was quantified by cross-correlation analysis.

The significance of possible changes in cycle periodduring deletions was determined with statistical analysisas described by Lafreniere-Roula & McCrea (2005).Cycle periods during deletions were compared with fivecycle intervals immediately preceding the deletion. Thet statistic (Lafreniere-Roula & McCrea, 2005) was used todecide whether the deletion cycle period was changed. Therunning average (Y2) and variance (s2) of X-cycle intervalswith sample size of n2 (n2 = 5) immediately before thedeletion were calculated, where X was the hypothesizednumber of cycles present during the deletion (e.g. X = 2for a single burst deletion). The deletion duration (Y 1)

was compared with the average of X-cycle intervals (Y2).The t statistic for the deletion was calculated according tothe following formula:

t = Y1 − Y2 − (μ1 − μ2)

s2 ·√

n2 + 1

n2

,

whereμ1 – μ2 is the hypothesized difference (μ1 – μ2 =0).A significance level of P < 0.05 with degrees of freedomn2 – 1 was used to determine whether the cycle periodduring the deletion was not different from the pre-vious cycles, and hence the deletion was defined as anon-resetting deletion; otherwise, the deletion was calleda resetting deletion.

To compare the synaptic drive to an identified neuronwith the ventral root output, we integrated the area fromthe neuron’s voltage trace above the baseline (minimalvoltage) during each cycle and compared it to theintegrated area over the same time period under thecorresponding smoothed and rectified ventral root burst.Since the units for the integrated neuronal voltage andventral root activity were different, we normalized theindividual values to the maximal values measured inthe eight cycles just before and just after the deletion,respectively. The normalized values for the neuronal andventral root activity were plotted against one another, andthe statistical significance of their relation was assessedusing linear regression analysis in Origin 8.

Modelling and simulations

Our model was developed using as a basis the earlierspinal locomotor CPG model of Rybak et al. (2006a); theoriginal model simulated locomotor rhythm generation inthe immobilized decerebrate cat during fictive locomotionevoked by electrical stimulation of the brainstem midbrainlocomotor region (MLR). The two-level structure of theCPG in each hemicord and the architecture of networkinteractions below the rhythm generator (RG) level, i.e.between ipsilateral populations representing the patternformation (PF) networks and populations of Ia inter-neurons, Renshaw cells and motoneuron populations,are the same in the present model as in the precedingmodel (Rybak et al. 2006a). The major changes in thepresent model include: (i) inclusion of two symmetricalcircuits representing the left and right CPGs, interactingvia commissural interneurons (CINs) synapsing at therhythm generator (RG) level; (ii) the RG on each sidehas an intrinsically rhythmic flexor half-centre and anon-rhythmic extensor half-centre; and (iii) there is no‘supra-spinal drive’ imitating MLR stimulation; instead,fictive locomotor activity is initiated by the elevated neuro-nal excitability that simulates the effect of applicationof NMDA and 5-HT evoking rhythmic locomotor-like



activity in the isolated spinal cord. The architecture of themodel is described in detail in Results, and the equationsimplementing the model and model parameters are givenin Appendix.

All neurons were modelled in the Hodgkin–Huxleystyle. Interneurons were simulated as single-compartmentmodels. Motoneurons had two compartments: somaand dendrite. As in the preceding Rybak et al. (2006a)model, we used the two-compartment motoneuron modelproposed by Booth et al. (1997). All interneuron types weremodelled as in the Rybak et al. (2006a) model. Howeverthe model parameters were adjusted to (a) make the flexorRG interneuron population on each side to be intrinsicallybursting, with frequency of oscillations within the rangethat seen during fictive locomotion evoked in the isolatedneonatal mouse spinal cord by NMDA and 5HT, and (b)elevate excitability of interneurons (defined by the leakreversal potential) so that the locomotor rhythm could begenerated without supra-spinal drive imitating the MLRstimulation.

Each neuron type in the model was representedby a population of 50–200 neurons. Heterogeneity ofneurons within each population was set by a randomdistribution of neuronal parameters and initial conditionsto produce physiologically realistic variations of base-line membrane potential levels, calcium concentrations,and other parameters of slow channel kinetics. A fulldescription of the model and its parameters can befound in the Appendix. All simulations were performedusing simulation package NSM 4.0 developed at DrexelUniversity by S. N. Markin, I. A. Rybak and N. A.Shevtsova. Differential equations were solved using theexponential Euler integration method with a step size of0.1 ms.

Results

Deletions of motor activity during NMDA/5-HTinduced fictive locomotion

Hindlimb locomotor-like activity (fictive locomotion)was induced in the isolated neonatal mouse (P0–P3)spinal cord preparation (n = 66) by bath applicationof a mixture of 8 μM NMDA and 8 μM serotonin(5-HT). Both wild-type ICR and Chx10::eCFP spinal cordswere used; the Chx10::eCFP strain has no observablebehavioural or electrophysiological phenotype (Zhonget al. 2010), and fictive locomotion was identical inboth strains. Fictive locomotion was characterized bymotoneuron burst alternation between the ipsilateraland contralateral segmental ventral roots, and betweenipsilateral flexor-dominated (L2) and extensor-dominated(L5) ventral roots on each side of the cord. The bursts ofmotoneuron activity from the extracellular ventral rootrecordings were rectified and smoothed, showing peaks of

activity corresponding to each burst (Fig. 1). We observedat least one spontaneous deletion episode during recordedfictive locomotion from 45 of the 66 preparations.

Two examples of flexor deletions seen in recordingsfrom the iL2 are shown in Fig. 1Aa and b. During thesedeletions, the flexor motoneurons failed to fire, as seen bythe absence of the expected peaks of activity (within theareas marked by grey bars) in the smoothed and rectifiedrecordings from the ventral root. During these deletions,the ipsilateral extensor root (iL5) fired continuously

Figure 1. Flexor and extensor deletions duringNMDA/5-HT-induced fictive locomotionAll records show smoothed and rectified traces of motoneuronactivity recorded extracellularly on the indicated ipsilateral (iL2, iL5)and contralateral (cL2, cL5) lumbar ventral roots. L2 recordings showpredominantly flexor motoneuron activity, while L5 recordings showpredominantly extensor activity. The bars above each set of tracesshow the expected timing of the bursts if the rhythm wereunperturbed during the deletion. Aa and b, two examples ofnon-resetting flexor deletions recorded from the L2 root (indicatedby the light grey bars) accompanied by tonic activity in the ipsilateralextensor (L5) root, but with no obvious effects on flexor or extensoractivity in the opposite side of the cord. Ba and b, multiplenon-resetting extensor and flexor deletions from two different spinalcords. Extensor deletions are indicated with dark grey bars, while thesingle flexor deletion is indicated with a light grey bar. C, anon-resetting flexor deletion occurred simultaneously in ipsilateral L2and L3 ventral roots, with no effect on contralateral L2 activity. D,the single example of simultaneous deletions on both sides of thecord. A flexor iL2 deletion occurs simultaneously with an extensorcL5 deletion. The iL2 deletion is accompanied by tonic activity in theiL5 ventral root, while cL2 rhythmic bursting is not perturbed duringthe cL5 deletion. See text for details.



throughout the period when the iL2 failed to burst, anddid not show the interruptions in firing normally observedduring the ipsilateral flexor bursts.

Figure 1Ba and b shows examples of extensor deletions,in which extensor bursts (three consecutive deletions inFig. 1Ba and one in panel Bb) were missing in the activity ofthe ipsilateral extensor root (iL5). During these deletions,the corresponding ipsilateral flexor activity (recorded fromthe iL2 ventral root) retained its normal cyclic bursting.

Most spontaneous deletions in the neonatal mousespinal cord are non-resetting

Each deletion identified during fictive locomotion wasanalysed to determine whether it was of a resettingor non-resetting type, using the statistical techniqueof Lafreniere-Roula & McCrea (2005) as described inMethods. In all, 216 episodes of locomotor activitycontaining deletions were recorded from 45 spinal cords.Of these, 92% (192) exhibited an integer number ofmissing cycles (P > 0.05) and were characterized asnon-resetting. Examples of such deletions are shownin Fig. 1, where the expected cycle timing is shownin the bars above each set of traces. In each case,in the ventral root recording showing a deletion, itresumed bursting at the expected time an integer numberof cycles later. The remaining 8% of deletions (24)occurred with rhythm resetting on both sides of thecord; many of these were obtained from cords withirregular locomotor bursting patterns and could reflect theirregularity of the bursting pattern rather than being trueresetting deletions. As a result, we focused our analysison non-resetting deletions in this paper (an exampleof a resetting deletion is described later in the paper).The percentage of non-resetting deletions in the neonatalmouse spinal cord was larger than that found in the cat(64%; Lafreniere-Roula & McCrea, 2005), suggesting thatit is more difficult to perturb the rhythm-generating kernelin the isolated neonatal mouse spinal cord than in thedecerebrate adult cat in vivo.

Asymmetry in antagonist motoneuron activity duringipsilateral flexor and extensor deletions

Of the 216 deletions found in our recordings, 56%(121) were flexor deletions while 44% (95) were extensordeletions. There was a marked asymmetry in the motoractivity of antagonist motoneurons during flexor vs.extensor deletions. During all of the flexor deletions(missing bursts in the activity of the iL2 ventral root), theipsilateral extensor root (iL5) exhibited sustained activitywith no interruptions at the times of the missing flexorbursts (see Fig. 1Aa and b). Similar results were seenin the remaining 119 episodes with flexor deletions. In

contrast, during all extensor deletions (missing burstsin the activity of the iL5 root), the ipsilateral flexorroot (iL2) continued unperturbed bursting (see Fig. 1Baand b). Figure 1Ba shows an example of a non-resettingdeletion of three sequential bursts in the iL5 extensorroot. Despite this long extensor silence, the iL2 flexorroot continued bursting, with unaltered cycle frequencyand burst amplitude. Similar results were seen in theremaining 93 episodes with extensor deletions. Note thatFig. 1Ba also shows a non-resetting flexor deletion in theactivity of the contralateral flexor root (cL2) (highlightedby the light grey bar). As expected, this flexor deletionwas accompanied by sustained activity in the contralateralextensor root (cL5). In four preparations, we recorded theactivity from both the iL2 flexor root and the adjacent iL3ventral root, which normally also carries predominantlyflexor motoneuron axons. In all four cases, a deletion inthe iL2 ventral root was also seen in the iL3 ventral root;an example is shown in Fig. 1C. These results suggest thatdeletions occur simultaneously in motor activity acrossmore than one spinal segment.

Spontaneous non-resetting motor deletions on oneside of the cord are independent of contralateralmotor activity

The locomotor phase maintenance followingnon-resetting flexor and extensor deletions, as wellas the persistence of ipsilateral flexor bursting duringextensor deletions and the sustained extensor activityduring flexor deletions, reflects local circuit interactionswithin the ipsilateral hemicord. However, the activity ofthese local circuits could also be affected by inputs fromthe contralateral hemicord. We tested this possibility inseveral ways.

First, we recorded from the L2 and L5 ventralroots on both sides of the cord, as shown in Fig. 1.These experiments showed that almost all spontaneousnon-resetting deletions (except for the single case inFig. 1D and described below) occurring on one side of thecord were not accompanied by disturbances in rhythmicactivity on the opposing side. Thus in Fig. 1Aa and b,deletions of flexor bursts in the iL2 root are accompaniedby sustained activity in the ipsilateral iL5 roots, but thereare no marked changes in activity recorded from thecontralateral cL2 or cL5 roots. Similarly, extensor deletionsin Fig. 1Ba and b are not accompanied by disturbancesin ipsilateral rhythmic flexor activity and also do notshow disturbances in contralateral flexor and extensorbursting. Of the 192 episodes with non-resetting deletions,only one broke this rule. This single case is shown inFig. 1D, where a flexor deletion in the ipsilateral flexorroot (iL2) occurred simultaneously with an extensordeletion in the contralateral extensor (cL5) root. Both



deletions occurred without a significant phase shift afterthe deletion (P > 0.05) and hence were formally classifiedas non-resetting. It is interesting to note that the rulesfor relations of ipsilateral flexor–extensor activity duringthese deletions were followed in each side even in thisexceptional case: the iL2 flexor deletion was accompaniedby sustained activity in the iL5 extensor root, while cL2flexor bursting was not perturbed during the cL5 extensordeletion. A possible explanation for this exceptionaldeletion is given in Discussion. All other spontaneousnon-resetting deletions in our experiments were limitedto one side of the cord and were not accompanied bydisturbances of rhythmic activity on the other side.

To further test the idea that non-resetting motordeletions on one side of the cord are not dependenton input from the contralateral cord, we recordedrhythmic motor activity from isolated left or righthemicords (n = 11). Fictive locomotion in an isolatedhemicord was induced by NMDA and 5-HT (Bonnotet al. 2002; Falgairolle & Cazalets, 2007), though moretime was required to stabilize the hemicord rhythmicbursting, and it had a much longer cycle period(around 10 s) than intact spinal cord preparations. Weobserved 15 episodes of deletions from seven hemi-cord preparations. Similar to the intact cord, 60% ofthe deletions (9 of 15) exhibited a failure of flexoractivity while 40% were classified as typical extensordeletions; all of these were non-resetting deletions. Inthese isolated hemicord preparations, the same rules ofipsilateral flexor–extensor interactions during the deletionwere observed. Specifically, burst deletions seen in theflexor root were accompanied by sustained activity inthe extensor root, while extensor deletions were notaccompanied by disturbances of rhythmic flexor activity.Figure 2A shows a non-resetting flexor deletion in the L2root (P > 0.25) accompanied by sustained activity in theL5 extensor root. Figure 2B shows a hemicord recordingwith three deletions. The first flexor deletion in the L2root was accompanied by sustained activity in the L5extensor root; the two subsequent extensor deletions seenin the L5 root were not accompanied by disturbances of L2bursting.

To identify the minimal region of the cord that cangive rise to these deletions, we recorded activity from iso-lated single L2 hemisegments (n = 5). With these hemi-segments, a longer time of perfusion with NMDA/5-HT(more than 1 h) was needed to evoke stable rhythmicactivity, and the cycle period was very slow (tens ofseconds; see Fig. 2C). We observed six episodes of deletionsfrom three single L2 hemisegments which expressed stablerhythmic activity. The deletion shown in Fig. 2C is anon-resetting flexor deletion, as seen by the tick marksand confirmed by our statistical analysis (P > 0.1). Theother five deletions were also non-resetting. We did notobserve deletions of more than one cycle in isolated hemi-

segments. These results show that the CPG ‘clock’ canbe functional within an isolated hemisegment, and thatthe resumption of activity an integer number of cycleslater does not require input from other parts of the spinalcord.

Motoneuron activity during deletions

To begin searching for the origins of deletions withinthe spinal locomotor networks during fictive locomotion,we used whole cell recordings to monitor the activityof flexor-related motoneurons during NMDA and 5-HTinduced fictive locomotion. The identity of the L2flexor-related motoneurons was verified by a collisiontest of orthodromic action potentials evoked by therecording electrode in the soma and antidromic actionpotentials evoked by stimulation of the ipsilateral L2ventral root (data not shown). During fictive locomotion,the activity of the paired left and right L2 ventral rootswas monitored. We made intracellular recordings from23 motoneurons during fictive locomotion, and detecteddeletions from 12. During ipsilateral ventral root deletions,

Figure 2. Motor deletions recorded from isolated spinalhemicords and hemisegmentsA, a flexor (L2) deletion (light grey bar) during rhythmic activity in thehemicord is accompanied by tonic activity in the L5 extensor root. B,multiple flexor and extensor deletions in a hemicord. The flexor (L2)deletion (light grey bar) is accompanied by tonic extensor (L5)activity, while two extensor deletions (dark grey bars) are notaccompanied by perturbation of rhythmic flexor bursting. C, anon-resetting flexor deletion (light grey bar) during NDMA and 5-HTinduced rhythmic activity from a single L2 hemisegment.



motoneurons appeared to lose most of their rhythmicsynaptic drive and did not fire action potentials. Oneexample during a non-resetting deletion (P > 0.2) isshown in Fig. 3A. As seen in this figure, rhythmic changesin synaptic drive to the motoneuron are in phase withthe bursts recorded from the ipsilateral ventral root.We analysed the relationship between the motoneuron’ssynaptically driven rhythmic depolarization during burstsand the corresponding ventral root burst output byintegrating the areas under each motoneuron oscillationand the smoothed and rectified ventral root activity,as described in Methods. There was a positive relationbetween the ventral root area and membrane oscillationarea (n = 16; r = 0.32; P = 0.031; Fig. 3B), showing thatas the drive to the recorded motoneuron increased,the summed output of the motoneurons recorded onthe ventral root also increased. During the deletion,the ventral root area fell close to zero; in parallel, themotoneuron membrane potential became flat and itsintegrated area also fell close to zero (Fig. 3B). Analysis ofintracellular recordings from 11 other flexor motoneuronsduring 18 episodes containing flexor deletions showedsimilar positive relationships between the integratedmotoneuron area and the integrated ventral root area(r = 0.36 ± 0.09; n = 12 neurons), with loss of synapticdrive during an ipsilateral non-resetting flexor deletion.Simultaneous recordings from ipsilateral and contra-lateral flexor roots demonstrated that rhythmic ipsilateralventral root activity and the rhythmic synaptic drive to

Figure 3. Loss of synaptic drive to flexor motoneurons duringnon-resetting flexor deletions during NMDA/5-HT-inducedrhythmic activityA, a flexor motoneuron loses its rhythmic depolarization and fallssilent during a non-resetting flexor deletion (grey bar). B, there is asignificant positive correlation between the integrated ipsilateralventral root activity during a burst and the integrated membranepotential oscillation of the motoneuron. During the ventral rootdeletion, the motoneuron displays almost no oscillation. The deletionsite is shown as the larger open circle. C, rhythmic synaptic drive andfiring of a different flexor motoneuron is unaffected during threecontralateral cL2 ventral root deletions.

the ipsilateral motoneuron were not perturbed duringcontralateral spontaneous flexor deletions. For example,in Fig. 3C, the cL2 ventral root showed three deletions,indicated by the grey bars. No corresponding disturbanceswere seen in the rhythmic iL2 activity or in the rhythmicpattern of synaptic drive to the iL2-related motoneuron.Similar results were seen in six motoneurons during 10locomotor episodes with deletions recorded in the contra-lateral cL2 ventral root. These results suggest that flexordeletions result from a perturbation in ipsilateral premotorcircuits, which can greatly reduce or eliminate the synapticdrive to flexor motoneurons. We have not yet performedparallel experiments with extensor-related motoneurons.

Activity of commissural interneurons duringlocomotor deletions

Commissural interneurons (CINs) send their axons tothe opposite side of the cord and coordinate left–rightalternation (Butt et al. 2002; Butt & Kiehn, 2003;Quinlan & Kiehn, 2007). We recorded CIN activityusing blind patch recordings along with iL2 and cL2ventral root recordings during NMDA/5-HT-inducedfictive locomotion. As found previously (Butt et al. 2002,2003; Zhong et al. 2006a,b; Quinlan & Kiehn, 2007), a sub-set of the CINs received strong rhythmic synaptic drive togenerate rhythmic bursts of action potentials during fictivelocomotion. We performed intracellular recordings fromsix highly rhythmic CINs during locomotor episodes withat least one deletion in the ipsilateral flexor iL2 activity. Fiveof these CINs received predominantly rhythmic excitatorysynaptic inputs which induced membrane oscillations inphase with the iL2 during fictive locomotion; thus theywere flexor-related CINs. One such example is shown inFig. 4A. This CIN showed rhythmic membrane potentialoscillations with occasional action potentials in phase withrhythmic iL2 root activity. The grey area from Fig. 4Ais expanded in Fig. 4B, showing a flexor deletion inthe iL2 ventral root. The oscillations in this CIN wereunperturbed during the deletion in the ipsilateral ventralroot. We measured the integrated locomotor burst areasfor both ventral root and intracellular CIN recordings.Unlike the motoneurons, we did not find any relationbetween these areas (n = 16; r = 0.03; P = 0.51; Fig. 4C).In particular, when the ventral root integrated area fellnear to zero during the deletion, the normalized CINarea was unaffected. Similar results were seen in fourother CINs during a total of 11 ipsilateral deletions(r = 0.04 ± 0.02 for correlation between integrated CINarea and integrated ventral root area; n = 4 neurons). Thesixth CIN received predominantly rhythmic inhibitoryinput; we did not perform detailed analysis on this singleneuron.



Two classes of V2a interneurons identified bydifferent behaviour during locomotor deletions

We next studied the activity of the genetically definedV2a interneurons during locomotor deletions. TheseChx10-expressing, ipsilaterally projecting, glutamatergicneurons are thought to provide synaptic drive to CINs(Al-Mosawie et al. 2007; Lundfald et al. 2007; Croneet al. 2008) and play important roles in maintainingleft–right alternation at high frequencies (Crone et al.2008, 2009; Zhong et al. 2010, 2011). Some V2a inter-neurons may also provide excitatory drive to ipsilateralmotoneurons (Lundfald et al. 2007; Crone et al. 2008;Dougherty & Kiehn, 2010). About half of V2a inter-neurons are rhythmically active during fictive locomotion(Dougherty & Kiehn, 2010; Zhong et al, 2010). In thisstudy, we recorded the activity of 19 rhythmic L2 V2ainterneurons which fired in phase with the iL2, each duringfictive locomotion episodes containing at least one flexordeletion in the iL2 ventral root activity. Depending ontheir activity during ipsilateral ventral root deletions, theseV2a neurons fell into two distinct groups. A majority ofV2a interneurons (n = 11), which we called type I V2ainterneurons, continued to oscillate regularly during 16episodes of deletions (Fig. 5). In contrast, the remainingeight V2a interneurons, which were called type II V2ainterneurons, lost their synaptic drive and showed nomembrane oscillations during 11 episodes of deletions(Fig. 6).

Figure 4. Activity of a flexor-related commissural interneuron(CIN) during a non-resetting flexor deletionA, the CIN shows rhythmic membrane potential oscillations in phasewith the iL2 root activity, and continues to oscillate during flexordeletion. B, enlargement of the grey area in A, showing the flexordeletion (light grey bar). C, there is no significant relation betweenthe integrated iL2 ventral root activity and the integrated membranepotential oscillation of the CIN. The deletion is indicated by thelarger open circle.

Figure 5A shows an intracellular recording from atypical type I V2a interneuron during a fictive locomotionepisode including a non-resetting ipsilateral flexordeletion (P > 0.2). This neuron’s activity was in phase withthe ipsilateral flexor root, iL2, and it continued to receiverhythmic synaptic drive and to fire rhythmically duringthe deletion. In this interneuron, there was no significantrelation between the integrated area of the extracellularventral root bursts and the interneuron’s integratedmembrane potential; the synaptic drive causing the voltageoscillation was not reduced during the iL2 deletion(n = 16; r = 0.02; P = 0.62; Fig. 5B). Five other neuronsclassified as type I V2a interneurons fired regularly duringthe deletions and did not show a relationship between theintegrated neuron area and the integrated ventral rootarea during bursting (r = 0.03 ± 0.01; n = 6 neurons).Thus, the synaptic input to this V2a interneuron andits activity were unaffected by the network perturbationproducing the ipsilateral flexor motoneuron deletion. Wealso recorded the synaptic currents in this interneuronusing voltage clamp recording at −45 mV (Fig. 5C); likemost V2a interneurons, this neuron was driven primarilyby rhythmic excitatory synaptic input which was in phasewith the iL2 activity (Dougherty & Kiehn, 2010; Zhonget al. 2010). During two iL2 flexor root deletions, thesynaptic drive to this V2a interneuron was not changed,demonstrating that the presynaptic input to this inter-neuron was not affected during the iL2 deletion. Figure 5Dshows the activity of another type I V2a interneuronduring a locomotor episode including a non-resettingflexor iL2 deletion; again the rhythmic synaptic drive tothis V2a neuron and its activity were unaffected duringthe deletion. After washout of NMDA and 5-HT to stopfictive locomotion, both this neuron and the iL2 ventralroot became quiescent. However, there were occasionalspontaneous prolonged bursts (up to 10 s in duration)of motoneuron activity recorded from the iL2 ventralroot (Fig. 5E; see also Hanson & Landmesser, 2003).During these spontaneous, non-locomotor ventral rootbursts, the type I V2a interneuron did not receive anysynaptic drive, and was silent. This result, combined withthe type I V2a neuron’s unperturbed rhythmic activityduring locomotor-related flexor burst deletions, impliesthat V2a interneurons of this type are not likely to bethe major class of pre-motor interneurons projectingto motoneurons, and instead may be components ofthe locomotor rhythm generator. Five other neuronsclassified as type I V2a interneurons were also silent duringnon-locomotor spontaneous ventral root bursts.

In contrast to the type I V2a interneurons, the type IIV2a interneurons exhibited a loss of activity in phase withipsilateral motoneuron deletions. All neurons of this typerecorded in our study received rhythmic synaptic driveand fired action potentials in phase with the flexor-relatedipsilateral L2 root. During iL2 deletions, these neurons



Figure 5. Rhythmic type I V2a interneurons continue to show oscillations and fire rhythmic bursts ofaction potentials during non-resetting flexor deletions occurring during NMDA/5-HT-induced fictivelocomotionA, a flexor-related V2a interneuron continues to oscillate and fire rhythmically during a flexor iL2 deletion. B, thereis no significant relation between the integrated iL2 ventral root activity and the integrated membrane oscillationsof the V2a interneuron. The deletion is indicated by the larger open circle. C, voltage clamp recordings showthat this V2a interneuron continues to receive rhythmic excitatory synaptic inputs during two non-resetting iL2deletions. D, another example of continued rhythmic activity of a type I V2a interneuron during an ipsilateral flexor(iL2) deletion. E, the type I V2a interneuron from D does not receive any synaptic drive during a spontaneous,non-locomotor iL2 burst.

not only were silent but also lost all rhythmic presynapticdrive. For example, the type II V2a interneuron in Fig. 6Afired rhythmically in phase with flexor iL2 ventral rootactivity. During a non-resetting flexor deletion (P > 0.2),

this interneuron lost its rhythmic synaptic drive, and fellsilent. Unlike the type I V2a interneurons, the integratedvoltage oscillation area of this interneuron correlatedpositively with the integrated area of the extracellular

Figure 6. Type II flexor-related V2a interneuronsfall silent and lose synaptic drive during flexordeletions during NMDA/5-HT-induced fictivelocomotionA, this V2a interneuron lost its rhythmic synaptic driveduring an iL2 flexor deletion. B, there is a significantpositive correlation between the integrated iL2 ventralroot burst amplitude and the integrated membranepotential oscillations of the V2a interneuron from A.The deletion is indicated by the larger open circle. C,the V2a interneuron in A is excited and fires aprolonged burst of action potentials during aspontaneous non-locomotor iL2 burst of activity. D,another example of a flexor-related type II V2ainterneuron that loses synaptic drive and falls silentduring a non-resetting flexor (iL2) deletion.



ventral root bursts (n = 16; r = 0.34; P = 0.022; Fig. 6B).During the iL2 deletion, this interneuron fell silent andits integrated voltage oscillation area fell close to zero,in parallel with the integrated ventral root burst area. Asimilar loss of synaptic drive was recorded from six othertype II V2a interneurons during motor deletions; theseneurons all showed a significant positive relation betweentheir integrated area and the integrated ventral root areaduring locomotor bursts (r = 0.35 ± 0.07; n = 7 neurons).Another example of a type II V2a interneuron losingsynaptic drive during an iL2 deletion is shown in Fig. 6C.After washout of NMDA and 5-HT, fictive locomotionceased, and type II V2a interneurons fell silent. However,during rare non-locomotor spontaneous ventral rootbursts, these neurons showed strong activation correlatedwith the motoneuron activation in the iL2 root (Fig. 6D).Thus, the type II V2a interneurons appear to comprise adistinct V2a population which has similar firing patternsto motoneurons during locomotor deletions, and mayindeed be pre-motor interneurons (Dougherty & Kiehn,2010).

Activity of V2a neurons during deletions of brainstemstimulation-evoked fictive locomotion

All the deletions described above occurred during fictivelocomotion evoked by bath application of NMDA and5-HT. Such pharmacologically evoked locomotor-likeactivity may be considered non-physiological, since itresults from non-specific and tonic activation of all NMDAand 5HT receptors in the spinal cord. We needed to makesure that the neuronal behaviours during deletions werenot artefacts of the NMDA/5-HT application. To testthis, we monitored the activity of interneurons duringfictive locomotion evoked by tonic electric stimulationof the brainstem (BES; Zaporozhets et al. 2004).BES-evoked fictive locomotion was also characterized byspontaneous deletions similar to those observed duringpharmacologically induced fictive locomotion. Over 90%of these deletions were non-resetting, as we foundwith pharmacologically evoked fictive locomotion. Wemonitored the activity of five rhythmic V2a interneuronsduring BES-evoked fictive locomotion: three of theseneurons were classified as type I V2a interneurons whiletwo were typical type II V2a interneurons. Figure 7A showsan example of a type I V2a interneuron recorded duringa BES-evoked fictive locomotion episode containing anon-resetting flexor deletion in the iL2 root. The activityof this neuron and its synaptic drive were not affectedduring the flexor deletion. As expected for a type I V2ainterneuron, there was no significant relation between theintegrated areas of the V2a membrane voltage oscillationand the extracellular ventral root bursts (n = 16; r = 0.09;P = 0.27; Fig. 7B). Similar results were seen for the other

two type I V2a interneurons during BES-induced fictivelocomotion. The type II V2a interneurons lost theirsynaptic drive and fell silent during ipsilateral iL2 deletions(data not shown). We conclude that the behaviour ofboth types of V2a neurons is independent of the methodby which fictive locomotion was produced, and reflectsthe structural organization of the CPG and the specificroles these interneuron types play in the locomotor CPGnetwork.

Activity of unidentified spinal interneurons duringlocomotor deletions

During our blind patch experiments, we occasionallyrecorded the activity of unidentified rhythmically activespinal interneurons during fictive locomotion. Werecorded the activity of 12 unidentified neurons whichwere not CINs, which were rhythmically active in phasewith the iL2 flexor motoneurons. During ipsilateral iL2deletions, 7 of these 12 neurons continued to displayrhythmic activity. Figure 8A shows an example of onesuch neuron during a non-resetting deletion. This neuroncontinued to oscillate and fire action potentials whenthe iL2 fell silent. As with the CINs and type I V2ainterneurons, this neuron’s integrated membrane voltagearea showed no significant relation to the integratedventral root burst areas (n = 16; r = 0.042; P = 0.45;Fig. 8B). Voltage clamp recordings at −45 mV showedthat this neuron was synaptically driven by inwardsynaptic currents in phase with ipsilateral ventral bursts,

Figure 7. Type I V2a interneurons continue to fire rhythmicallyduring non-resetting deletions occurring during BES-inducedfictive locomotionA, a flexor-related type I V2a interneuron continues to receiverhythmic synaptic drive and fire action potentials during an iL2deletion. B, there is no significant relation between the iL2integrated ventral root amplitude and the integrated membranepotential oscillations of the V2a interneuron.



and outward currents during contralateral ventral rootbursts (Fig. 8C). Two non-resetting ipsilateral flexor iL2deletions occurred in this recording, one of which lastedfor five consecutive cycles. Both the rhythmic excitatoryand inhibitory phases of synaptic drive to this neuronwere unaffected during these deletions, and the cyclefrequency remained constant (Fig. 8C). The remainingfive unidentified interneurons lost their synaptic drive andfell silent during iL2 motor deletions; their firing patternwas similar to the ipsilateral motoneurons and the type IIV2a interneurons (data not shown).

A two-level asymmetrical model of the locomotorCPG

We have generated a model of the mouse neonatallocomotor CPG to help interpret our experimental results.Figure 9A shows a schematic diagram of our model ofa hemicord locomotor CPG. This model was developedusing a combination of two basic concepts: the concept oftwo-level CPG organization, as previously implementedin the Rybak–McCrea model (Rybak et al. 2006a,b;McCrea & Rybak, 2007, 2008), and the Duysens–Pearsonconcept of an asymmetric rhythm generator with adominant flexor half-centre or with a pure flexor-relatedrhythm generator (Pearson & Duysens, 1976). Similar tothe Rybak–McCrea model, the present model has twofunctional/hierarchical levels referred to as the ‘rhythmgenerator’ (RG) and ‘pattern formation’ (PF) networks.To implement the Duysens–Pearson concept of aflexor-dominant locomotor generator, which is implicitlysuggested by our experimental data showing asymmetricaleffects of flexor and extensor deletions (Fig. 1), the RGin the current model has only one intrinsically rhythmichalf-centre generating a flexor-related rhythmic activity

(RG-F population). As in the previous Rybak et al.(2006a) model, bursting in this population is based ona persistent (slowly inactivating) sodium current in eachneuron and mutual excitatory synaptic interactions withinthe RG-F population (the existence of this current inthe spinal cord was confirmed by several studies, seeZhong et al. 2007; Tazerart et al. 2008; Brocard et al.2010; Ziskind-Conhaim et al. 2010). The non-rhythmicextensor half-centre (RG-E population) is tonically active.However, its activity contributes to control of the durationof the extensor phase and the timing of switching tothe next flexion via the inhibitory In-RG-E population,whose activity is in turn regulated by the rhythmic RG-Fhalf-centre via the inhibitory In-RG-F population. Inaccordance with the two-level concept, the RG networkin this model performs a ‘clock’ function: it defines thelocomotor frequency, coordinates left and right rhythmicpatterns (via different types of CINs), and drives theactivity of the PF network.

The PF network has two principal populations, PF-Fand PF-E, which drive locomotor activity in flexor andextensor motoneuron populations, respectively. Thesepopulations reciprocally inhibit each other via the In-PF-Fand In-PF-E populations and drive alternating activity inthe various flexion- and extension-related populations ofthe ipsilateral locomotor network. The PF-F populationreceives rhythmic drive from the RG-F rhythm generatorand provides rhythmic drive to the flexor motoneurons.The PF-E population receives tonic drive from the RG-Epopulation, and is rhythmically inhibited during the flexorphase by the PF-F neurons via the inhibitory In-PF-Fneurons. The PF-E population provides rhythmic driveto the extensor motoneurons, and can help regulate PF-Factivity via the inhibitory IN-PF-E interneurons.

We have expanded this model to reflect interactionsbetween CPG networks on the left and right sides of the

Figure 8. Responses of unidentified neuronsduring deletions occurring duringNMDA/5-HT-induced fictive locomotionA, an unidentified non-commissural interneuroncontinues to fire rhythmically during an iL2 deletion. B,there is no significant relation between the integratediL2 ventral root burst amplitude and this neuron’snumber of action potentials per cycle. The deletion siteis indicated by the larger open circle. C, voltage clamprecording of synaptic inputs to the neuron from Aduring multiple iL2 deletions. The rhythmic synapticdrive to the neuron is maintained during the deletions.



spinal cord. The schematic diagram of the full model thatincorporates interactions between symmetrical circuits inthe left and right hemicords is shown in Fig. 9B. The leftand right CPGs (each organized as shown in Fig. 9A) inter-act at the RG level via CIN populations (Butt & Kiehn,2003). Specifically (see Fig. 9B) the l-CINi-F and r-CINi-Fpopulations provide mutually inhibitory interactionsbetween left and right flexor RG half-centres (l-RG-F andr-RG-F); the l-CINe-F and r-CINe-F populations provideexcitatory inputs from each RG flexor half-centre (l-RG-Fand r-RF-F) to both contralateral RG half-centres (r-RG-Fand r-RG-E, and l-RG-F and l-RG-E, respectively); andthe l-CINe-E and r-CINe-E populations provide excitatoryinput from each extensor half-centre (l-RG-E and r-RG-E)to contralateral flexor half-centres (r-RG-F and l-RG-F,respectively). In addition, inhibitory interactions betweenipsilateral flexor and extensor antagonists operate via theinhibitory Ia-F and Ia-E populations, which also inhibiteach other and receive inhibition from the correspondingpopulations of Renshaw cells (R-F and R-E), whose activityshapes firing activity of the corresponding motoneuronpools. Organization of these lower-level interactions isbased on a large body of data obtained in previous studiesin the cat (McCrea et al. 1980; Lundberg, 1981; Shefchyk& Jordan, 1985; Pratt & Jordan, 1987; Jankowska, 1992;Geertsen et al. 2011) and was used in the earlier Rybaket al. (2006a) model.

Simulation results

The performance of our model under normalconditions and during simulated perturbations producingnon-resetting deletions is shown in Fig. 10. In accordancewith the two basic concepts described above, afictive locomotor-like pattern with alternating ipsilateralflexor–extensor and left–right bursting activity isgenerated in the model by two (left and right) rhythmicflexor half-centres (l-RG-F and r-RG-F) and is coordinatedby the interactions between left and right half-centresvia the inhibitory and excitatory CIN populations. Thearchitecture of neuronal interactions implemented inthe model (Fig. 9B) allows it to generate coordinatedflexion-related oscillations at the RG level, and trans-form these oscillations (through the specific interactionsbetween different neuron populations on each sideof the cord) into the left–right and flexor–extensoralternating locomotor-like activity of the correspondingmotoneuron populations. The integrated activity of allmajor populations is shown in Fig. 10.

As an initial test of the functional roles of theCIN-mediated interactions between left and right circuitsin the model, we simulated the effect of hemisection,i.e. a full removal of all contralateral circuits and CINpopulations. The result of this simulation is shown

in Fig. 11. This figure shows that removal of contra-lateral circuits in the model reduced the frequencyof locomotor oscillation, approximately by half, whilemaintaining the symmetric ipsilateral flexor–extensoralternation (approximately equal durations of flexor andextensor phases). These simulations closely reproduceour experimental data on the results of hemisection(see Fig. 2A and B and compare with records inFig. 1). Our analysis has shown that the reductionin locomotor frequency after simulated hemisectionresults mainly from the elimination of excitatory inputto the rhythm-generating ipsilateral RG-F populationfrom the contralateral RG-E population, which maybe considered as a prediction for further experimentalstudies.

We then focused our study on the changes in inter-neuron and motoneuron rhythmic activity during variousdeletions, as described by our experimental studies. Asindicated in Fig. 9A, deletions could be produced inthe model by simulated disturbances evoked by eitherapplication of short-duration excitatory or inhibitorysignals to particular PF or RG populations or temporarysuppression of inputs to these populations. In oursimulations, non-resetting flexor and extensor deletionson each side could be produced by simulated perturbationsaffecting the model at the level of the ipsilateralPF circuits. Examples of simulated perturbations thatproduce non-resetting deletions are shown in Fig. 10.The first deletion in this figure was produced by abrief suppression of excitatory input from the left RG-E(l-RG-E) to the left PF-E population (l-PF-E), as shown inFig. 9A by the open arrow. Application of this perturbationis indicated by the bar at the top of the figure and bythe dark grey rectangle. This perturbation produced anon-resetting deletion in the activity of the left extensormotoneurons (l-Mn-E, second trace) without disturbingrhythmic activity of the ipsilateral flexor motoneurons(l-Mn-F, top trace) or the CINs and contralateral flexoror extensor motoneurons (two bottom traces). Thiscorresponds to the non-resetting extensor motor deletionsobserved in our experimental studies (Fig. 1Ba and b).The second disturbance in Fig. 10 (illustrated by thelight grey rectangle and the bar at the bottom ofthe figure) demonstrates a non-resetting flexor deletionoccurring on the right side of the cord (two flexor burstsare missed in activity of the right flexor motoneuron,r-Mn-F). This non-resetting deletion was produced bya brief suppression of excitatory input from the rightRG-F (r-RG-F) to the right PF-F population (r-PF-F),as shown in Fig. 9A by another unfilled arrow. Similarnon-resetting flexor deletion could be also producedby a brief increase of activity (e.g. by an externalexcitatory drive) to r-PF-E, which would then inhibit ther-PF-F population. This non-resetting flexor deletion wasaccompanied by sustained activity of ipsilateral (right)



Figure 9. Schematic diagram of the modelPopulations of interneurons are represented by spheres. Excitatory and inhibitory synaptic connections are shown byarrows and small circles, respectively. Populations of motoneurons are represented by diamonds. A, the two-levelasymmetrical model of the locomotor CPG generating and controlling rhythmic activity in one hemicord. TheCPG consists of a rhythm generator (RG) and a pattern formation (PF) network. The asymmetrical RG has twohalf-centres: an intrinsically rhythmic flexor half-centre (RG-F population, red sphere), generating flexor-relatedrhythmic activity, and a tonically active flexor half-centre (green sphere), interacting via inhibitory interneuronpopulations (In-RG-E and In-RG-F). The PF network contains interneuron populations (PF-F and PF-E, green spheres).



Figure 10. Performance of the model andsimulation of non-resetting deletionsActivities of all excitatory populations and CINs in theleft and right hemicords are shown. Activity of eachpopulation (in this and following figures) is representedby the average histogram of neuronal activity in thecorresponding population (number of spikes per secondper neuron, bin = 50 ms). The colour of each tracecorresponds to the population type (see Fig. 9). Themodel generates coordinated flexion-related oscillationsin each RG and transforms these oscillations into theleft–right and flexor–extensor alternating locomotor-likeactivity of the corresponding motoneuron populations.This simulation shows two non-resetting deletionepisodes indicated by the grey rectangles. The firstdeletion episode represents a non-resetting deletion ofleft extensor activity accompanied by rhythmic flexoractivity (dark grey rectangle). This deletion wasproduced by a brief removal of excitatory input to thel-PF-E population from the l-RG-E (as shown in Fig. 9Aby an open arrow). The applied perturbation is indicatedby the bar at the top of the traces. The second deletionepisode represents a non-resetting deletion of rightflexor activity accompanied by tonically active extensors(see light grey rectangle). This deletion was produced bya brief removal of excitatory input to the r-PF-Fpopulation from the r-RG-F (shown in Fig. 9A by theother open arrow). Application of this perturbation isindicated by the bar at the bottom of the traces. Duringboth deletion episodes the activity of the contralateralpopulations is unaffected. See text for details.

extensor motoneurons (second trace from the bottom)without any disturbances in the rhythmic activity of CINsand contralateral flexor or extensor motoneurons (two toptraces). This corresponds to the non-resetting flexor motordeletions observed in our experimental studies (Figs 1Aaand b and 4).

As described above, non-resetting deletions could beproduced in the model by perturbations applied to the PFnetwork on one side of the cord, i.e. below the ipsilateralRG circuits and CIN populations. As a consequence, it isnot surprising that these perturbations did not disturbrhythmic activity of the CINs and contralateral inter-

neurons and motoneurons. This is consistent with ourexperimental data showing no effect of non-resettingdeletions on CINs and contralateral motoneurons (Figs 3Cand 4, respectively).

Although we did not observe many unambiguousexamples of resetting deletions in our experiments, suchresetting deletions can occur, and have been analysed inthe cat (Lafreniere-Roula & McCrea, 2005). Our modelcan produce resetting deletions, shifting the phase of thefollowing motor bursts, by a perturbation that affectsthe circuitry at the RG level. Figure 12 shows examplesof such simulations using our model. In Fig. 12A, the

that receive inputs from the corresponding RG populations and inhibits each other via the inhibitory populations(In-PF-E and In-PF-F). Possible sources of perturbations producing various resetting and non-resetting deletions areindicated by different arrows (see key). B, schematic diagram of the full locomotor model with bilaterally organisedlocomotor CPGs interacting via populations of commissural interneurons (CINs). The model has symmetricallyorganized left and right networks with the identical CPG circuits on each side organized as shown in A. Thenetwork in each side also includes Ia interneuron populations (Ia-F and Ia-E), Renshaw cells (R-F and R-E), andflexor and extensor motoneuron populations (Mn-F and Mn-E). Prefix l- or r- in the population names indicatesthat the population belongs to the left or right hemicord, respectively. See text for details.



applied perturbation, shown at the top (bar), temporarilyinhibited the rhythmic l-RG-F population, producingan ipsilateral deletion in flexor motoneurons (l-Mn-F).As in case of non-resetting deletions, this motoneuronflexor deletion was accompanied by sustained activityof ipsilateral extensor motoneurons (l-Mn-E). However,in this example the deletion was resetting (see thephase shift indicated at the bottom of the motoneurontraces). Although the perturbation was applied only tothe ipsilateral l-RG-F population, it also affected contra-lateral activity via the CIN populations projecting to thecontralateral RG populations. As a result, the appliedperturbation also disturbed the activity of contralateralmotoneurons (two bottom traces). Specifically, during

Figure 11. Simulation of hemisectionA, activity of the principal RG populations and motoneuronpopulations in the intact model. B, changes in the activity of thesame populations after simulated hemisection (removal of all circuitslocated in the right hemicord). See text for details.

the deletion of the ipsilateral l-RG-F flexor burst, thispopulation did not inhibit the contralateral flexor burstin r-RG-F via the l-CINi-F population (see Fig. 9B). Inaddition, the sustained activity of l-RG-E during thisdeletion additionally excites the contralateral r-RG-F (seeFig. 9B), producing an advanced onset of contralateralflexor busts in r-RG-F and in the r-Mn-F population,and a partial deletion (reduction in both amplitudeand duration) of the right extensor r-Mn-E motoneuronburst. This disturbance also produced some transitionalpost-perturbation temporal dynamics before returning tothe stable oscillations, with an obvious phase shift relativeto the initial rhythm.

The simulation in Fig. 12B was specially performed tofit a rare experimentally recorded episode with a clearlyresetting deletion, shown in Fig. 12C. In this experiment,regular bursting activity was recorded before and after theresetting deletion. The deletion episode in this experimentis characterized by (a) a partial extensor deletion on theipsilateral side (in the iL5 root: dark grey bar) and (b)a flexor deletion on the contralateral side (in cL2: lightgrey bar) that was accompanied by sustained activity inthe associated extensor root (cL5). Figure 12B reproducessuch a resetting deletion in our model by a brief inhibitionapplied to the l-RG-E population, shown at the top (bar).This simulated perturbation produced both an ipsilateralextensor deletion (see l-Mn-E) and a contralateral flexordeletion (see r-Mn-F) accompanied by sustained activityin the contralateral extensors (see r-Mn-E). Specifically,suppression of ipsilateral extensor activity in l-RG-Eremoved inhibition from this population to the ipsilateralflexor half-centre (l-RG-F, via the inhibitory l-In-RG-Epopulation, see Fig. 9B), thus producing an advancedonset of the next ipsilateral flexor burst in l-RG-F. Thisburst, via the commissural l-CINi-F, strongly inhibitedthe contralateral (right) flexor RG half-centre (r-RG-F,see Fig. 9B), finally causing the flexor deletion of thecontralateral flexor motoneurons (r-Mn-F) accompaniedby a sustained activity in right extensor motoneurons(r-Mn-E). As indicated in Fig. 12B, and similar to thecorresponding experimental episode in Fig. 12C, thesedeletions shift the phase of subsequent bursts and thusare of the resetting type.

Discussion

Spontaneous deletions in rhythmic locomotor activityin the neonatal mouse spinal cord

Analysis of spontaneous motor deletions duringMLR-evoked fictive locomotion in the decerebrate,immobilized cat showed that about two-thirds of deletionswere non-resetting, suggesting that they did not resultfrom perturbations in the rhythm-generating (RG) kernelof the CPG (Lafreniere-Roula & McCrea, 2005; Rybak et al.



Figure 12. Simulation of resetting deletionsA, rhythm disturbance was produced by a brief inhibition of the l-RG-F population (indicated by a bar abovethe traces). This perturbation produced a resetting flexor deletion in the left flexor motoneuron activity (l-Mn-Fpopulation) accompanied by a sustained activity in left extensor motoneurons (l-Mn-E population) (marked by alight grey rectangle). Although applied to the left RG, this perturbation also affected the activity of the contralateral(right) RG (via commissural interneurons, not shown) and finally perturbed the activity of right extensor and flexormotoneurons (r-Mn-F and r-Mn-E populations) (marked by a dark grey rectangle). As indicated in the figure, theapplied perturbation produced a phase shift after resumption of rhythmic motoneuron activity in both sides. B, inthis example the rhythm was disturbed by a brief inhibition of the l-RG-F population (shown as a bar at the top).This perturbation produced an ipsilateral partial deletion in the extensor motoneurons (see l-Mn-E trace) (indicatedby dark grey rectangle) and a contralateral flexor deletion on the right side (see r-Mn-F trace) accompanied bysustained extensor activity (r-Mn-E trace) (marked by the light grey rectangle). Rhythmic activity resumed withan identical phase shift on both sides. This simulation was specifically performed to reproduce the experimentalepisode of a resetting deletion shown in panel C below. C, an experimentally recorded episode with a resettingdeletion. An unknown perturbation produced a partial extensor deletion on the ipsilateral side (in the iL5 root) anda flexor deletion on the contralateral side (in cL2) that was accompanied by sustained activity in the extensor cL5.In all panels (A–C), the horizontal bars with vertical ticks located above and below motoneuron traces indicate thetiming of locomotor periods before and after deletions, allowing the visualization of the phase shift between theinitial and resumed rhythms. See text for details.



2006a). Our experiments in the isolated neonatal mousespinal cord show that the percentage of non-resettingdeletions (92%) is even larger than in the decerebrate cat.This suggests that the RG network in this preparation ismore resistant to resetting than in the adult cat in vivo. Thisdifference does not appear to result from different methodsused to evoke fictive locomotion. While most of ourexperiments with the neonatal mouse spinal cord evokedfictive locomotion by application of NMDA and 5-HT, wefound the same high frequency of non-resetting deletionsduring BES-evoked fictive locomotion. The different agesand species of the animals (adult cat vs. neonatal mouse)might explain the greater resistance to locomotor rhythmresetting in the neonatal mouse.

Our results show that in the isolated neonatal mousespinal cord, spontaneous locomotor deletions on oneside of the cord are normally not accompanied byrhythm disturbances on the opposite side. Non-resettingdeletions can occur in an isolated hemicord, showingthat maintenance of the rhythm phase during thedeletion does not require inputs from the opposite sideof the cord. Our results confirm that each hemicordcontains an independent rhythmogenic network that canfunction in the absence of the other hemicord, althoughthe left and right networks are normally coupled viacommissural interneurons. We have also shown thatlocomotor oscillations with non-resetting deletions occureven in an isolated hemisegment. This confirms thedistributed nature of the rhythmogenic components ofthe CPG, as previously shown by lesion studies (Cazaletset al, 1995; Kjaerulff & Kiehn, 1996; Cowley & Schmidt,1997; Kiehn & Kjaerulff, 1998).

Firing properties of spinal neurons during motordeletions

The neonatal mouse spinal cord preparation offers aunique opportunity to monitor the activity of identifiedclasses of neurons during locomotor deletions. Werecorded the activity of motoneurons and two typesof identified interneurons thought to be involved inlocomotor pattern generation, V2a interneurons andCINs, during locomotor deletions. As expected, the firingactivity and integrated membrane potential oscillationsof motoneurons projecting to the ipsilateral ventral rootscorrelated with the root’s integrated activity; deletions ofventral root activity always accompanied a loss of synapticdrive and failure to fire in the corresponding motoneurons(Fig. 3). This suggests that the last-order interneuronsproviding the synaptic drive to motoneurons also fall silentduring deletions.

Our experiments reveal the existence of two functionalsubtypes of V2a interneurons with different behavioursduring ipsilateral flexor deletions. Rhythmic activity in the

type I V2a interneurons was not perturbed during flexordeletions in the ipsilateral ventral root. The integratedsynaptic drive to these neurons did not correlate with theintegrated motor output, and did not decrease during theventral root deletion. In addition, the type I V2a inter-neurons remained silent during spontaneous ipsilateralventral root bursts in the absence of fictive locomotion. Wesuggest that the type I V2a interneurons do not providedirect synaptic drive to motoneurons, but are insteadinvolved (directly or indirectly) in rhythm generationand/or coordination between left and right networks viathe CINs. This class could, for example, constitute the V2ainterneurons that form anatomically defined synapses onthe V0 CINs (Crone et al. 2008).

In contrast to type I V2a interneurons and similarto motoneurons, the type II V2a interneurons fellsilent during non-resetting ipsilateral flexor deletions.Moreover, they lost locomotor related synaptic driveduring these deletions, and their integrated membranepotential oscillations correlated positively with theintegrated bursts of the corresponding ipsilateral ventralroot. In addition, they fired at high frequency duringspontaneous ipsilateral ventral root bursts in the absenceof fictive locomotion. These results suggest that the type IIV2a interneurons do not belong to the RG circuits (whoseactivity is not perturbed during non-resetting deletions),but can be components of the pattern formation networkand/or last-order interneurons that directly project tomotoneurons (Lundfald et al. 2007; Crone et al. 2008;Dougherty & Kiehn, 2010). The loss of synaptic drive tothese neurons during non-resetting deletions suggests thatthe higher order interneurons which drive them also fallsilent during these deletions.

The commissural interneurons (CINs), which sendtheir axons contralaterally and coordinate phaserelationships between left and right locomotor networks,have been shown to display a wide variety of firingproperties during fictive locomotion (Butt et al. 2005;Butt & Kiehn, 2003; Jankowska et al. 2003, 2005; Zhonget al. 2006a,b; Quinlan & Kiehn, 2007). We found thatall flexor-related rhythmic CINs recorded in our studycontinued to receive rhythmic synaptic input and tofire rhythmically during non-resetting ipsilateral flexordeletions. This supports the idea that these CINs receivesynaptic drive from the ipsilateral rhythm generator,which continues its rhythmic activity during non-resettingdeletions. Some of these CINs may also receive excitationfrom the type I V2a interneurons (Crone et al. 2008).

A two-level asymmetrical model of the neonatalmouse locomotor CPG

The phenomenon of non-resetting deletions during fictivelocomotion in the cat has previously been explainedby a model of a two-level locomotor CPG controlling



each limb, which contains a top-level RG, maintainingthe period and phase of locomotor oscillations, and alower-level PF network operating under control of theRG and driving the activity of motoneuron populations(Rybak et al. 2006a, McCrea & Rybak, 2007). Accordingto this model, reciprocal inhibition between antagonisticcircuit elements in each hemicord operates at both RGand PF levels. Perturbations at the RG level cause resettingmotor deletions, while perturbations at the PF level causenon-resetting motor deletions (Rybak et al. 2006a, McCrea& Rybak, 2007). This earlier model, however, did notconsider contralateral left–right interactions. To supportbilateral interactions, three types of commissural neurons(CIN populations) have been incorporated in our model(see Fig. 9B). Such excitatory and inhibitory CINs havebeen described experimentally (Cowley & Schmidt, 1995;Butt & Kiehn, 2003; Hinckley et al. 2005; Quinlan &Kiehn, 2007), and a subset of the CINs receive strongrhythmic synaptic drive to generate rhythmic activityduring fictive locomotion (Butt et al. 2002, 2003; Zhonget al. 2006a,b; Quinlan & Kiehn, 2007). In our currentbilateral two-level model, the commissural interneuronsmediate left–right interactions only at the RG level,i.e. between left and right RG interneuron half-centrepopulations. If this organization is correct, then inter-neurons that are involved in generation of the locomotorrhythm should demonstrate rhythmic activity duringfictive locomotion that should not be perturbed duringnon-resetting deletions. This could include neurons thatbelong to the left or right RG, CINs that coordinate inter-actions between the left and right RGs, and the type I V2ainterneurons that could transfer RG drive to the CINs.In addition, however, there may be other types of CINswhich are presumably involved in the operation at the PFcircuits, which may directly project to motoneurons, as hasbeen previously described (Butt & Kiehn, 2003; Quinlan& Kiehn, 2007). The activity of such CINs is expected to beperturbed during non-resetting deletions. However, suchCINs have not been detected in our current study.

One of the major results of this work is that wehave indeed found both types of identified interneurons:those that are presumably involved in the operation ofRG on each side or in interactions between left andright RGs, and those which instead operate at the PFlevel. Specifically, the CINs and type I V2a interneuronsrecorded in our study have the features of interneuronsinvolved in RG operation described above, while thetype II V2a interneurons appear to fit to the descriptionof the PF level interneurons. They may belong to PFcircuits or represent last-order interneurons organizingthe phasing and activity of the corresponding motoneuronpopulations. In addition, during blind patch recordings,we monitored the activity of 12 other unidentified spinalinterneurons which were rhythmically active during fictivelocomotion; 7 of these neurons showed no perturbation

during non-resetting motor deletions (suggesting arelation to RG network activity), whereas the remainingfive interneurons lost activity during the non-resettingmotor deletions (suggesting a relation to PF networkactivity). All of these results provide support for theconcept of a two-level CPG organization.

Another striking result of our current study of theneonatal mouse isolated spinal cord preparation was thefinding of strong asymmetry in motor activity duringspontaneous ipsilateral flexor and extensor deletions.Specifically, during non-resetting flexor deletions theipsilateral extensor root always showed sustained firing,whereas rhythmic flexor activity was never disturbedduring non-resetting deletions of extensor activity.This may reflect a fundamental asymmetry in theorganization of the neonatal mouse hindlimb locomotorCPG, which was previously suggested in studies offictive and treadmill locomotion in cats (Pearson &Duysens, 1976, Duysens & Pearson, 1980; Whelan,1996; Wilson et al. 2005; Duysens, 2006; Brownstone& Wilson, 2008). The adult decerebrate cat duringMLR-evoked fictive locomotion exhibited more deletiontypes (Lafreniere-Roula & McCrea, 2005). Both flexor andextensor deletions could be accompanied by sustainedactivity in the antagonist nerves or muscles. However,while extensor deletions with unaffected flexor burstingwere described in these and other cat locomotion studies,there were no clear examples in which flexor deletionswere accompanied by unperturbed extensor bursting.This observation indirectly supports the so-called ‘flexorburst generator model’ originally suggested by Pearson& Duysens (1976), who hypothesized an asymmetriclocomotor CPG in which a flexor rhythm generator isthe dominant rhythm-generating half-centre (see alsoDuysens, 2006; Guertin, 2009). Our computational modelcombines the Rybak–McCrea concept of the two-levellocomotor CPG (Rybak et al. 2006a,b; McCrea & Rybak,2007, 2008) with the Duysens–Pearson concept of anasymmetric rhythm generator with a dominant flexorhalf-centre (Pearson & Duysens, 1976). Our experimentalresults are in full accordance with the Duysens–Pearsonhypothesis, so in our current model, the RG layer includesonly one intrinsically rhythmic half-centre which drivesrhythmic flexor activity (the RG-F population). Basedon the previous Rybak et al. (2006a) model, burstingin this population is based on a persistent (slowlyinactivating) sodium current, present in all RG-F neurons,and mutual excitatory synaptic interactions within theRG-F population. The non-rhythmic extensor half-centre(the RG-E population) is tonically active. However, viainhibitory interactions with the intrinsically rhythmicflexor half-centre it can control the duration of theextensor phase, and the timing of switching to the nextflexion. In addition, as we demonstrated in simulation(Fig. 11), excitatory interactions of this half-centre with



the contralateral RG-F, via specific excitatory CINs, mayspeed up locomotor oscillations; this can explain theexperimentally observed slowing of the locomotor cyclefrequency in the spinal cord after hemisection (Fig. 2A).

The proposed computational model was able toreproduce both types of non-resetting deletions observedin our experimental studies by inhibition of specificneuron classes in the model (Fig. 10). In addition, weused the model to simulate resetting perturbations anddeletions (Fig. 12). These simulations emphasized that theimportant feature of resetting deletions (distinguishingthem from non-resetting deletions) is the disturbance ofrhythm and a corresponding post-deletion phase shift onboth sides of the cord. Our model was able to reproduce thedetails of motoneuron activity during one experimentalresetting deletion (compare Fig. 12B and C). In thisconnection, it is interesting to re-consider the exceptionaldeletion shown in Fig. 1D, when a flexor deletion in theipsilateral flexor root (iL2) occurred simultaneously withan extensor deletion in the contralateral extensor (cL5)root. Because there was no phase shift after these deletions,they were formally classified as non-resetting. However,because rhythmic pattern disturbances were seen on bothsides of the cord (which is typical of the resetting deletiontype), it is possible that this episode represents a resettingdeletion occurring with a zero phase shift. Note that thedisturbances/deletions produced in this episode in all fourventral roots are qualitatively similar to those shown inFig. 12C (though the sides of the cord are switched), whichsuggests that they are produced by qualitatively similarnetwork perturbations such as those modelled in Fig. 12B.While the episode in Fig. 12C was resetting because itclearly exhibited a phase shift of locomotor oscillationsafter the deletions, this phase shift was insignificant in theepisode shown in Fig. 1D. This suggests that deletion inFig. 1D represents a ‘degenerate case’ of resetting deletionsoccurring with a zero phase shift.

Our model was built based on our experimental data ofspontaneous deletions from the isolated neonatal mousespinal cord preparation; it may not reflect CPG networkorganization in adult mice, or in more intact in vivo pre-parations. We believe that intrinsic properties of someCPG interneurons, and synaptic interactions betweenthem, may change during postnatal maturation (forexample see Abbinanti & Harris-Warrick, 2008; Huschet al. 2011), which could alter the function of the networkas the animal gets older. Also, sensory feedback canaffect the locomotor CPG at several levels (modelledby Rybak et al. 2006b; McCrea & Rybak, 2008) andmodify their operation during real locomotion. Finally,deletions evoked by other means, such as pharmacologicalmanipulation of synaptic strength (Talpalar & Kiehn,2010), may affect network function in different ways fromthe spontaneous deletions we have studied, and provideadditional insights into network organization.

Conclusion

The concept of a two-level CPG organization containingindependently controlled rhythm-generating (RG) andpattern-forming (PF) networks was based on many linesof evidence (Perret & Cabelguen, 1980; Kriellaars et al.1994; Guertin et al. 1995; Perreault et al. 1995; Rybaket al. 2006a,b; McCrea & Rybak, 2007, 2008). Ourwork in the neonatal mouse spinal cord confirms thisearlier work, and, for the first time, provides electro-physiological recordings of identified interneurons (V2aand CINs) during non-resetting deletions predicted bythe model for RG and PF neurons. These recordings haveclearly demonstrated the flexor–extensor asymmetry inthe neonatal mouse locomotor CPG. The challenge forthe future is to place these interneurons more firmly inthe network structure and show how they contribute torhythmogenesis and locomotor pattern formation. Ourmodel makes testable predictions of the synaptic drivethat neurons at each point in the CPG should receiveunder control conditions and during different types ofdeletions; this should provide important informationto assign functional roles to these and other identifiedinterneurons.

Appendix

Modelling of single motoneurons and interneurons

In accordance with the two-compartment motoneuronmodel of Booth et al. (1997), but with the additionalincorporation of the persistent sodium current (INaP)to the motoneuron dendrite, the membrane potentialsof the motoneuron soma (V (S)) and dendrite (V (D)) aredescribed by the following differential equations:

C · dV(S)

dt= −INa(S) − IK(S) − ICaN(S) − IK,Ca(S)

− IL(S) − IC(S);

C · dV(D)

dt= −INaP(D) − ICaN(D) − ICaL(D) − IK,Ca(D)

− IL(D) − IC(D) − ISynE − ISynI, (A1)

where C is the membrane capacitance and t is time andindexes (S) and (D) indicate ionic currents in the someand dendrite, respectively.

The dendrite–soma coupling currents (withconductance gC) for soma IC(S) and dendrite IC(D)

are described following the Booth et al. (1997) model:

IC(S) = g C

p· (V(S) − V(D));

IC(D) = g C

1 − p· (V(D) − V(S)), (A2)



Table 1. Maximal conductances of ionic channels and EL in different neuron types

Neuron type gNa gNaP gK gCaL gCaN gK,Ca gL EL∗

(nS) (nS) (nS) (nS) (nS) (nS) (nS) (mV)

RG-F 30 0.35 2 0.09 −64 ± 0.64RG-E 30 0.1 2 0.09 −60 ± 0.6PF 30 0.1 2 0.09 −62 ± 0.62All other interneurons 120 100 0.51 −64 ± 3.2Mn soma 120 100 14 2 0.51 −68 ± 3.4Mn dendrite 0.1 0.33 0.3 0.8 0.51 −68 ± 3.4

∗To provide heterogeneity of neurons within neural populations, the value of EL wasrandomly assigned from normal distributions (see mean values ± SDs).

where p is the parameter defining the ratio of somaticsurface area to total surface area.

The ionic currents in the model are described as follows:

INa = g Na · m3Na · hNa · (V − E Na);

INaP = g NaP · mNaP · hNaP · (V − E Na);

IK = g K · m4K · (V − E K);

ICaN = g CaN · m2CaN · hCaN · (V − E Ca);

ICaL = g CaL · mCaL · (V − E Ca);

IK,Ca = g K,Ca · mK,Ca · (V − E K);

IL = g L · (V − E L), (A3)

where V is the membrane potential; INa is the fastsodium with maximal conductance g Na; INaP is thepersistent sodium current with maximal conductanceg NaP; IK is the delayed-rectifier potassium current withmaximal conductance g K; ICaN is the N-type calciumcurrent with maximal conductance g CaN; ICaL is the L-typecalcium current with maximal conductance g CaL, IK,Ca

is calcium-dependent potassium current with maximalconductance g K,Ca; IL is the leakage current withconductance gL; ENa, EK, ECa, and EL are the reversalpotentials for sodium, potassium, calcium and leakagecurrent, respectively.

For simplicity and because of the lack of specific data,all interneurons (single-compartment models) except RGand PF neurons contain only a minimal set of ioniccurrents:

C · dV

dt= −INa − IK − IL − ISynE − ISynI . (A4)

The excitatory neurons of the CPG comprising theRG and PF populations have the persistent (slowlyinactivating) sodium current, INaP:

C · dV

dt= −INa − INaP − IK − IL − ISynE − ISynI. (A5)

However, only RG-F neurons have maximalconductance of this current (g NaP) large enough toactivate endogenous bursting properties in these neurons.

Maximal conductances of all ionic currents for allneuron types are listed in Table 1.

Activation m and inactivation h of voltage-dependentionic channels (e.g. Na+, NaP

+, K+, CaN2+, CaL

2+) aredescribed by the following differential equations:

τmi(V) · d

dtmi = m∞i(V) − mi ;

τhi(V) · d

dthi = h∞i(V) − hi,

(A6)

where i identifies the name of the channel, m∞i(V )and h∞i(V ) define the voltage-dependent steady-stateactivation and inactivation, respectively, and τmi(V ) andτhi(V ) define the corresponding time constants. Activationof the sodium channels is considered to be instantaneous(τmNa = τmNaP = 0). The parameters and expression for allchannel kinetics can be found in Table 2.

The kinetics of intracellular Ca2+ concentration (Ca,described separately for each compartment) is modelledaccording to the following equation (Booth et al. 1997):

d

dtCa = f · (−α · ICa − kCa · Ca), (A7)

where f defines the percentage of free to total Ca2+; αconverts the total Ca2+ current, ICa, to Ca2+ concentration;kCa represents the Ca2+ removal rate.

Activation of the Ca2+-dependent potassium channels isconsidered instantaneous and described as follows (Boothet al. 1997):

mK,Ca = Ca

Ca + K d, (A8)

where Ca is the Ca2+ concentration within thecorresponding compartment or neurone, and K d definesthe half-saturation level of this conductance.

Synaptic excitatory (ISynE with conductance gSynE

and reversal potential ESynE) and inhibitory (ISynI withconductance gSynI and reversal potential ESynI) currentshave also been incorporated into our models:

ISynE = g SynE · (V − E SynE);

ISynI = g SynI · (V − E SynI). (A9)



Table 2. Steady state activation and inactivation variables and time constants for voltage-dependent ionicchannels

Ionic m∞(V); V is in mV τm(V) (ms)channels h∞(V); V is in mV τh(V) (ms)

Na+ m∞Na = (1 + exp(−(V + 35)/7.8))−1 τmNa = 0h∞Na = (1 + exp((V + 55)/7))−1 τhNa = 30/(exp((V + 50)/15) + exp( − (V + 50)/16))

NaP+ m∞NaP = (1 + exp(−(V + 47.1)/3.1))−1 τmNaP = 0

h∞NaP = (1 + exp((V + 59)/10))−1 τhNaP = τhNaPmax/ cosh (V + 59)/20), τhNaPmax = 10000K+ m∞K = (1 + exp(−(V + 28)/15))−1 τmK = 7/(exp((V + 40)/40) + exp( − (V + 40)/50))

hK = 1CaN

2+ m∞CaN = (1 + exp(−(V + 30)/5))−1 τmCaN = 4h∞CaN = (1 + exp((V + 45)/5))−1 τhCaN = 40

CaL2+ m∞CaL = (1 + exp(−(V + 40)/7))−1 τmCaL = 40

hCaL = 1

All expressions and parameters, except for NaP+, are taken from Booth et al. (1997). The expressions for

the NaP+ channel are from Rybak et al. (2003).

The excitatory (gSynE) and inhibitory synaptic (gSynI)conductances are equal to zero at rest and may beactivated (opened) by the excitatory or inhibitory inputsrespectively:

g SynEi(t) = g E ·∑

j

S{wj i} ·∑tk j <t

exp(−(t − tk j )/τSynE)

+ g Ed ·∑

m

S{wdmi} · dmi ;

g SynIi(t) = g I ·∑

j

S{−wj i} ·∑tk j <t

exp(−(t − tk j )/τSynI)

+ g Id ·∑

m

S{−wdmi} · dmi, (A10)

where the function S{x}= x, if x ≥ 0, and 0 if x < 0.According to eqn (A10), the excitatory and inhibitory

synaptic conductances have two terms. The first termdescribes the integrated effect of inputs from other neuro-nes in the network (excitatory and inhibitory respectively).The second term describes the effect of external drives usedto simulate applied perturbations. Each spike arriving toneurone i from neurone j at time tkj increases the excitatorysynaptic conductance by g E · wj i if the synaptic weightwji > 0, or increases the inhibitory synaptic conductanceby −g I · wj i if the synaptic weight wji < 0. g E and g I arethe parameters defining an increase in the excitatory orinhibitory synaptic conductance, respectively, producedby one arriving spike at |wji| = 1. τSynE and τSynE are thedecay time constants for the excitatory and inhibitoryconductances, respectively. In the second terms of eqn(A10), g Ed and g Id are the parameters defining the increasein the excitatory or inhibitory synaptic conductance,respectively, produced by external input drive dmi = 1 witha synaptic weight of |wdmi| = 1. The weights of all synapticconnections between the neural populations are shown inTable 3.

Table 3. Weights of synaptic connections in the network

Target population∗Source population or drive (weight of

synaptic input to one neuron)

i-CINe-F i-RG-F (0.015)i-CINi-F i-RG-F (0.015)i-CINe-E i-RG-E (0.03)i-RG-F i-RG-F (0.0025) with probability 0.1;

i-In-RG-E (0.002); c-CINe-F (0.00006);c-CINi-F (−0.012); c-CINe-E (0.005)

i-RG-E i-RG-E (0.005); c-CINe-F (0.01)i-In-RG-F i-RG-F (0.015)i-In-RG-E i-RG-E (0.015); i-In-RG-F (−0.6)i-PF-F i-RG-F (0.003); i-In-PF-F (−0.0025)i-PF-E i-RG-E (0.018); i-In-PF-E (−0.13)i-In-PF-F i-PF-E (0.04); i-In-PF-E (−0.013)i-In-PF-E i-PF-F (0.04); i-In-PF-F (−0.013)i-Ia-F i-PF-F (0.06); i-Ia-E(−0.04); i-R-F (−0.04)i-Ia-E i-PF-E (0.06); i-Ia-F(−0.04); i-R-E (−0.04)i-R-F i-Mn-F (0.1); i-R-E (−0.02)i-R-E i-Mn-E (0.1); i-R-F(−0.02)i-Mn-F i-PF-F (0.035); i-Ia-E (−0.1);i-R-F (−0.04)i-Mn-E i-PF-E (0.035); i-Ia-F (−0.1);i-R-E (−0.04)

∗Prefixes i- and c- indicate ipsi- and contralateral populations.Values in brackets represent relative weights of synapticinputs from the corresponding source populations (wji).

Other neuronal parameters. General parameters: ENa =55 mV; EK = −80 mV; ECa = 80 mV; C = 1 μF cm−2;gC = 0.1 mS/cm2; p = 0.1; f = 0.01; α = 0.0009mol C−1 μm−1; kCa = 2 ms−1; K d = 0.2 μM.

Parameters of synapses: ESynE = −10 mV; ESynI =−70 mV; g E = 0.05 mS cm−2; g I = 0.05 mS cm−2;g Ed = 0.05 mS cm−2; g Id = 0.05 mS cm−2; τSynE = 5 ms;τSynI = 5 ms.



Modelling neural populations

Each type of neuron in the model was representedby a population of neurons. In all simulations in thispaper, RG-F population consisted of 200 neurons, RG-E,PF-F, PF-E and both MN populations consisted of 100neurons, and all other populations contained 50 neurons.Connections between populations were established suchthat if a population A was assigned to receive anexcitatory or inhibitory input from a population B orexternal drive D, then each neurone in the populationA received the corresponding excitatory or inhibitorysynaptic input from each neuron in the population Bor from drive D with a probability of connection PAB

or PAD, respectively (a random generator was used todefine the existence of each connection if PAB or PAD

was not equal to 1). For all interconnections betweenpopulations, except for those inside the left and rightRG-F (l-RF-F and r-RG-F) these probabilities were set to1. For mutual interconnections within l-RG-F and r-RG-Fpopulations, the probabilities of connections were set to0.1. Heterogeneity of neurons within each population wasset by a random distribution of leak reversal potentialEL in each neuron and initial conditions for all slowvariables. The latter were chosen randomly from a uniformdistribution for each variable, and a settling period of50 s was allowed in each simulation before data werecollected. Each simulation was repeated 10–15 times,and demonstrated qualitatively similar behaviour forparticular values of the standard deviation of EL and initialconditions.

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Author contributions

The experiments in this study were performed in the Departmentof Neurobiology and Behaviour, Cornell University, and themodelling and simulations were performed in the Department ofNeurobiology and Anatomy, Drexel University. G.Z. and R.H.W.conceived and designed the electrophysiological experiments.G.Z. collected and analysed the experimental data. N.S. andI.R. developed the model and conducted all simulations. G.Z.,N.S., I.R. and R.H.W. drafted and revised the article. All authorsapproved the final version for publication.

Acknowledgements

We thank Bruce Johnson, Andreas Husch and Shelby Dietz forimportant conversations and input to this manuscript, and VinayPatel and Connor Benton for assistance in data analysis. Thiswork was supported by NIH grants NS17323 and NS057599,and NSF grant IOS-0749467 to R. Harris-Warrick, and NIHgrant NS04884 to I. Rybak.

Author’s present address

G. Zhong: Department of Chemistry and Chemical Biology,Harvard University, Cambridge, MA, USA.


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