Locomotor Pattern Generation in the Rodent Spinal …pages.nbb.cornell.edu/neurobio/harris-warrick/148.pdf · Locomotor Pattern Generation in the Rodent Spinal Cord Ronald Harris-Warrick*

Locomotor Pattern Generation in the Rodent Spinal Cord

Ronald Harris-Warrick*

Department of Neurobiology and Behavior, Cornell University, Ithaca, NY, USA

Synonyms

CPG; Locomotor central pattern generator; Spinal locomotor network

Definition

The locomotor central pattern generator is a neural network in the spinal cord that can generate thebasic motor pattern for locomotion in the absence of sensory feedback or rhythmic input from thebrain. Most research in rodents has focused on hind limb movements: the hind limb CPG is locatedin lower thoracic and lumbar segments of the spinal cord. This network generates both thelocomotor rhythm (cycle frequency) and the detailed phasing of motoneuron activation duringthe cycle, including alternation of left and right limbmovements and alternation of ipsilateral flexorand extensor activity.

Detailed Description

A central pattern generator (CPG) is a limited neural network that can produce an organizedrhythmic motor output in the absence of sensory or descending inputs from other parts of thenervous system (Marder and Calabrese 1996). CPGs drive behaviors such as locomotion, respira-tion, mastication, and digestion (Gossard et al. 2010). The CPG for vertebrate locomotion is locatedin the spinal cord. This entry will focus on the organization of the rodent hind limb locomotor CPG,which directs rhythmic movements of the left and right hind limbs of rats and mice. Under normalcircumstances, in the intact animal, locomotor CPGs are strongly affected by sensory feedback,which can, for example, determine the timing of the swing phase initiation during walking, bydescending inputs from the brain, which normally provide tonic drive to activate the locomotorCPG, and by modulatory inputs which shape CPG output and prepare it for activation.

The rodent spinal cord is particularly useful for studies of CPG function because the locomotorCPG can be activated in the isolated spinal cord (Smith and Feldman 1987). Motor output ismonitored with extracellular recordings of motoneuron activity from the ventral roots (VRs);higher lumbar VRs (L1–2) contain axons predominantly from flexor motoneurons (MNs), whilelower lumbar VRs (L4–5) predominantly contain axons of extensorMNs (Fig. 1a). The CPG can beactivated by a number of methods which nonspecifically excite the isolated spinal cord. Theseinclude pharmacological manipulation (typically with a combination of N-methyl-D-aspartate(NMDA) and serotonin), sensory stimulation by tonic activation of dorsal roots, and tonic

*Email: [email protected]

Encyclopedia of Computational Neuroscience

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stimulation of the brainstem or specific activation of brainstem regions such as the parapyramidalregion (Jordan 1998; Jordan et al. 2008). The elicited motor output shows alternation of left andright segmental VRs (e.g., left and right L2) combined with alternation of ipsilateral flexor (L2) andextensor (L5) MN activity (Fig. 1b). This is the fictive equivalent of walking.

Lesion studies have shown that the rodent CPG is distributed continuously along the lowerthoracic and lumbar segments of the spinal cord (Kudo and Yamada 1987b; Kjaerulff and Kiehn1996; Cowley and Schmidt 1997). Each segment, and even each hemisegment, is capable ofgenerating a rhythmic output with appropriate MN phasing, showing that both the “clock,” orrhythm-generating component of the CPG, and the pattern-organizing network, which determinesthe phasing of MN activation, are widely and redundantly distributed over many spinal segments.Coordination of this distributed network requires ascending and descending interneuron coordi-

nating pathways, which have not yet been identified in rodents.

Identified Interneurons in the Mouse Spinal Locomotor CPGRecent studies of the developmental determination of spinal interneurons in mice have providedimportant tools to identify interneurons that may be components of the hind limb locomotor CPG(Stepien and Arber 2008; Goulding 2009; Gosgnach 2011; Kiehn 2011). These interneurons areidentified by selective expression of defining transcription factors during development, whichdrives differentiation of the interneuron subtypes from common precursors. Modern genetictechniques allow visualization of these neurons as well as manipulation of their activity andselective elimination. The major interneuron types implicated in locomotor CPG function aredescribed below.

V0 InterneuronsThis is a heterogeneous class of interneurons, defined by embryonic expression of Dbx1. Almost allof the V0 interneurons are commissural, sending their axons to synapse on neurons on the oppositeside of the spinal cord. As expected, they play a role in left-right hind limb coordination. The V0group has been subdivided into four subgroups based on subsequent transcription factor expressionand location of origin (Gosgnach 2011; Kiehn 2011). All of these groups are rhythmically activeduring fictive locomotion in the neonatal mouse spinal cord, suggesting that they may participate in

Fig. 1 Fictive locomotion in the isolated rodent spinal cord. (a) Experimental preparation, with extracellular

recordings from the right and left L2 (flexor-dominated) and L5 (extensor-dominated) ventral roots of the lumbar

spinal cord. Recordings from identified interneurons have been mostly performed in the rostral region. (b) simulta-

neous extracellular recordings from left and right flexor (L2) and the left extensor (L5) ventral roots during fictive

locomotion evoked by bath application of NMDA, serotonin (5-HT), and dopamine (DA). Alternation across the cord

(lL2 and rL2) and between ipsilateral flexors and extensors (lL2 and lL5) are seen



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the locomotor CPG. V0D interneurons comprise 70 % of the V0 class; they are predominantlyinhibitory, contralaterally projecting commissural interneurons (CINs). The V0V class representsthe majority of the remaining V0 neurons; they express Evx1/2 and are excitatory glutamatergicCINs. The V0C group (5 % of the class) expresses Evx1/2 and Pitx2 and generates cholinergicinterneurons that have either commissural or ipsilaterally directed axons; these neurons are thesource of the cholinergic C-boutons found on predominantly ipsilateral MNs (Zagoraiou et al.2009). There is also a small group of V0G neurons which are glutamatergic, V0V-derived, Pitx2-positive neurons (Zagoraiou et al. 2009). Ablation of the V0V, V0C, and V0G groups in Evx1mutant mice did not affect fictive locomotion (Lanuza et al. 2004); selective loss of cholinergicfunction in the V0C group does not affect behavioral locomotion but may selectively reduceextensor muscle activity during swimming behavior (Zagoraiou et al. 2009). Ablation of the entireV0 population in Dbx1 mutant mice leads to a disruption of left-right alternation, with driftbetween the two sides during neonatal fictive locomotion (Lanuza et al. 2004), and may enhancea hopping behavior in intact mice (Kiehn 2011). This emphasizes the major role of the commissuralV0 interneurons in coordinating the networks on the left and right sides of the spinal cord.

V1 InterneuronsThe V1 class is characterized by expression of En1 and encodes ipsilaterally projecting inhibitoryGABAergic/glycinergic interneurons. Only about 30% of these neurons have been physiologicallycharacterized. Renshaw cells are detected by their selective expression of En1 and calbindin. Theyare activated by motoneuron collateral axons and provide a negative feedback to inhibit themotoneurons that excite them. They are rhythmically active, in phase with either ipsilateral flexoror extensor motoneurons during fictive locomotion, and may help terminate MN activity in eachcycle (Nishimaru et al. 2006). They receive rhythmic excitatory drive from MNs, which releaseboth ACh and glutamate in the neonate (Nishimaru et al. 2005), and ipsilateral and contralateralrhythmic inhibition to shape their output. Another set of V1 neurons differentiates into Ia inhibitoryinterneurons; these are activated by muscle spindle afferents and monosynaptically inhibit antag-onist muscles. Flexor- and extensor-related Ia inhibitory interneurons are mutually inhibitory.

These neurons are rhythmically active during the inhibitory phase of the antagonist MNs. Deletionor acute silencing of the entire V1 population causes a dramatic slowing of the motor pattern andprolongation of motoneuron firing during the cycle, with little effect on the motoneuron phasing(Gosgnach et al. 2006). This suggests that the V1 population has input to the rhythm-generatingcomponents of the locomotor CPG, though how this is effected is unknown; presumably this is onefunction of the 70 % of V1 interneurons that are not yet identified.

V2 InterneuronsThe V2 class is derived from progenitors that express Lhx3 and contains several different subtypes.The V2a class expresses Chx10 and forms glutamatergic, excitatory ipsilaterally projectinginterneurons. V2a neurons have been shown to synapse onto Evx1-expressing V0V commissuralinterneurons and appear to play a role in regulation of left-right alternation (Crone et al. 2008).Some V2a neurons also synapse onto ipsilateral motoneurons. Genetic deletion of V2a interneu-rons has little motor phenotype at low locomotor frequencies, but as the frequency of walkingincreases, left-right alternation is much more variable than usual, and at high frequencies,a synchronous left-right bounding gait is observed which is never seen in control mice (Croneet al. 2008, 2009). A similar frequency-dependent effect is seen in the isolated spinal cord duringfictive locomotion. V2a interneurons are only weakly active at low frequencies but are increasinglyactive and recruited as the cycle frequency increases (Zhong et al. 2010). Thus, these neurons seem



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to play a major role to prevent a gait change to bounding at high cycle frequencies. Recent studiesof the consequences of spontaneous motor deletions during fictive locomotion (described below)suggest that the V2a interneurons can be split into two subgroups, one of which is related to therhythm generator network in the CPG and drives CINs, while the other is an output path from thepattern formation networks of the CPG and provides some of the drive to ipsilateral motoneurons(Zhong et al. 2012). The V2b class of interneurons expresses GATA2/3 and forms ipsilaterallyprojecting inhibitory interneurons. Little is known about this class, though it may form a smallsubset of the Ia inhibitory interneurons which are mostly generated from the V1 class (Kiehn 2011).A new class of V2c interneurons has recently been identified which are derived from the GATA3-expressing V2b lineage and express Sox1 (Panayi et al. 2010). At present, nothing is known of theirroles in the locomotor CPG.

V3 InterneuronsThe V3 class is derived from progenitors that express Sim1 (Zhang et al. 2008). These interneuronsare excitatory and glutamatergic; most of them are commissural, though about 15 % haveipsilateral axons. These neurons form synapses onto contralateral and ipsilateral motoneurons,Renshaw cells, and Ia interneurons. Selective silencing of V3 neurons caused a marked degener-ation in the regularity of the fictive locomotor rhythm both in vivo and in the isolated neonatalspinal cord: there were marked variability in the duration of ventral root bursts and asymmetry inleft and right bursting (Zhang et al. 2008). However, the basic rhythmic alternating motor patternwas conserved. Thus, V3 interneurons appear to be important in retaining the regularity of themotor rhythm, especially by coordinating left and right sides of the cord.

Hb9 InterneuronsThe Hb9 transcription factor is predominantly expressed in motoneurons, but it is also expressed ina small set of interneurons located adjacent to the central canal in the ventral cord. Theseinterneurons are rhythmically active during fictive locomotion and show endogenous oscillatoractivity when excited by NMDA and serotonin. This has led to suggestions that the Hb9 interneu-

rons may be components of the rhythmogenic kernel of the locomotor CPG (Hinckley et al. 2005;Wilson et al. 2005; Brownstone andWilson 2008; Brocard et al. 2010). However, during locomotoractivity, their onset of firing lags behind that of ipsilateral motoneurons; during electricallystimulated fictive locomotion, they are only weakly active or silent after the first few cycles,despite continued strong rhythmic motoneuron firing (Kwan et al. 2009). These results suggest thatthey may not be the sole rhythmic drive for locomotion, but they could participate in mediating therhythm generator network’s drive of lower-level interneurons and motoneurons.

dI6 InterneuronsThis mixed class of commissural and ipsilaterally projecting interneurons arises from Lbx1-espressing progenitors in the dorsal embryonic spinal cord and migrates ventromedially to laminaeVII/VIII, where the locomotor CPG is localized. Transmitter labeling suggests that these area mixture of excitatory and inhibitory interneurons. They have been proposed to express Wt1(Goulding 2009); an inhibitory subpopulation expresses Dmrt3 (Vallstedt and Kullander 2013),which may help identify them in future experiments. Physiological analysis of dI6 neurons (Dycket al. 2012) showed that the majority are rhythmically active during fictive locomotion. Physio-logically, they divide into two populations: one population possesses endogenous rhythmiccapability, while the other fires rhythmically due to synaptic stimulation. Dyck et al. proposethat the endogenously rhythmic neurons may be components of the rhythm-generating kernel of the



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CPG, along with other interneurons. Mutants lacking Dmrt3 show relatively normal fictivelocomotion at low speeds but increasing lack of coordination both of left-right and ipsilateralflexor-extensor phasing at higher speeds; Vallstedt and Kullander (2013) propose that they may actas phase-lock neurons to control and secure a robust gait.

Organization of the Segmental CPG for Locomotion in RodentsIn preparation for a computational model of the neonatal rodent locomotor CPG, it is useful to try toplace the identified interneurons into a context where they drive selected components of the motorpattern, including the mechanisms for rhythmogenesis, left-right hind limb coordination, andflexor-extensor alternation within a limb. Although these components have not been studied indetail using mathematical models, schematics have been developed which could serve as templatesfor future modeling efforts.

RhythmogenesisAs described in detail below, studies of the consequences of locomotor deletions during fictivelocomotion have implicated the existence of a separate rhythm-generating network within thelocomotor CPG, which provides the rhythmic drive for locomotion in the isolated spinal cord. Theneuronal composition of the rhythm-generating kernel remains unknown, despite considerablework. Blockade of inhibitory synapses with a combination of GABA and glycine antagonists(Cowley and Schmidt 1995), or targeted deletion of the V1 inhibitory interneurons (Gosgnach et al.2006), does not eliminate rhythmogenesis, though it can slow the rhythm frequency. A number ofresults suggest that ipsilaterally projecting glutamatergic neurons are essential for normalrhythmogenesis (Kiehn 2011); rhythmic activity can be generated by an isolated left or righthemicord and even an isolated hemisegment (Kudo and Yamada 1987a; Zhong et al. 2012).Blockade of glutamatergic synapses eliminates the rhythm, and optogenetic excitation ofglutamatergic neurons is sufficient to activate locomotor activity (Hagglund et al. 2010). However,the identity of these glutamatergic neurons is not known. Elimination of either of the knownglutamatergic interneurons, V2a and V3, affects the locomotor pattern but does not eliminate the

Fig. 2 Schematic of organization of the commissural system that coordinates left and right limb movements.

Excitatory interneurons and synapses are shown in red, while inhibitory interneurons and synapses are shown in

blue. Only one half of the network is shown, with identified neurons on the left making synapses onto neurons on the

right. See text for details (From Kiehn 2011)



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rhythm (Crone et al. 2008; Zhang et al. 2008; Crone et al. 2009); neither of these neurons hasintrinsic rhythmogenic properties. The Hb9 interneurons show endogenous rhythmicity and arerhythmically active during fictive locomotion; however, their weak and delayed activation duringthe locomotor cycle (trailing behind the ipsilateral motoneurons they are supposed to drive)suggests that they receive drive from other rhythmogenic neurons (Kwan et al. 2009). The dI6interneurons contain a subpopulation which is endogenously rhythmic and shows loosely coupledrhythmic firing beginning before the onset of ipsilateral MN activity (Dyck et al. 2012). Dyck et al.propose that these interneurons may participate in the rhythmic kernel of the locomotor CPG;selective inactivation of these neurons has not yet been accomplished. The rhythmogenic kernelcould activate rhythmic firing due to endogenous bursting properties of the kernel interneurons(Brocard et al. 2010), which may only become manifest during activity-dependent changes inextracellular ion concentrations (Brocard et al. 2013). Alternatively, a network of mutuallyexcitatory interneurons with spike frequency adaptation could form the kernel (Kiehn 2011).This network could arise from the combined interactions among several of the currently identifiedgroups (none of which is essential) or an entirely new group that has not yet been specified.

Left-Right Hind Limb CoordinationThrough their detailed studies of the commissural interneurons (CINs), Kiehn and colleagues havegenerated the most sophisticated model for the coordination of left-right limb movements (Fig. 2;Kiehn 2011). Electrophysiological measurements demonstrated the existence of three separatecommissural pathways to contralateral motoneurons (Butt and Kiehn 2003). A direct inhibitorypathway uses glycinergic CINs to monosynaptically inhibit contralateral MNs. A disynapticinhibitory pathway uses glutamatergic CINs to drive contralateral GABAergic and/or glycinergicinterneurons which inhibit MNs (Butt and Kiehn 2003; Quinlan and Kiehn 2007). This dualinhibitory commissural pathway could serve to maintain contralateral flexor (or extensor) MNsout of phase with ipsilateral flexor (or extensor) activity. A third pathway uses glutamatergic CINsto directly excite contralateral MNs. These could serve to couple activity of flexors with contra-lateral extensors; they also appear to coordinate synchronous left-right activity, as seen after

blockade of glycinergic synapses with strychnine (Cowley and Schmidt 1995), or in mutants thatreduce crossed inhibitory commissural projections (Kullander et al. 2003; Rabe et al. 2009).

In Fig. 2, the direct inhibitory pathway is proposed to be mediated by the V0D class of inhibitoryCINs, while the indirect inhibitory pathway is thought to be mediated by the excitatory V0V and V3commissural interneurons synapsing onto contralateral V1 interneurons, including Renshaw cells,Ia inhibitory interneurons, and other unidentified V1 neurons (Kiehn 2011). The monosynapticexcitatory pathway is proposed to be mediated by the V3 interneurons; in mutant Netrin-1�/�mice,the V3 interneurons are the major surviving commissural pathway, and the motor pattern issynchronous across the cord (Rabe et al. 2009). The V3 pathway could also help maintainalternation under normal conditions, as the flexor center on one side could use them to excite thecontralateral extensor MNs (Butt and Kiehn 2003). The rhythmic drive to these CINs has not beenfully elucidated. However, a subset of the excitatory V2a interneurons form synapses ontomembers of the V0V neurons and could help drive the indirect inhibitory commissural pathway(Crone et al. 2008). This is not the only drive to this pathway, as locomotor behavior and fictivelocomotion are essentially normal at low frequencies in mutants lacking these neurons; only athigher frequencies is left-right coordination disrupted, suggesting that other unidentified inputsprovide the predominant drive to V0V CINs at lower speeds (Crone et al. 2009). Based on detailedstudies of the spinal locomotor CPG in aquatic organisms, CINs are very likely to synapse on



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neurons in the rhythm-generating kernel of the CPG, as well as other interneurons in the network.This is indicated in Fig. 2 by the dotted lines to the rhythm center on the right side.

Flexor-Extensor AlternationThe third component of the locomotor pattern is alternation between ipsilateral flexor (F) andextensor (E) motoneurons (Fig. 3; Kiehn 2011). F-E alternation is preserved in isolated hemicordpreparations, suggesting that contralateral input, while it may support F-E alternation, is notessential (Kudo and Yamada 1987a; Zhong et al. 2012). Identified ipsilaterally projecting inhib-itory interneurons include the heterogeneous V1 population and the V2b neurons. In the V1population, the Renshaw cells mainly provide recurrent inhibition to motoneurons (Alvarez et al.2013). The Ia inhibitory interneurons mediate sensory crossed reflexes that can support F-Ealternation: flexor-related Ia-INs inhibit extensor-related Ia-INs and MNs, and vice versa. BothRenshaw cells and IA-INs are rhythmically active during fictive locomotion (Nishimaru et al.2006). Over 70 % of the V1 population remains unidentified. Genetic deletion of the entire V1population does not abolish F-E alternation, and some reciprocal inhibition is still detected in theabsence of the IA-INs (Gosgnach et al. 2006). The inhibitory GATA 2/3-expressing V2b inter-neurons could also participate in F-E phasing (Kiehn 2011). Finally, in the intact cord, CINs fromthe opposite side of the cord could help coordinate ipsilateral flexor-extensor alternation by bothexcitatory inputs (which could couple contralateral flexors and extensors) and inhibitory inputs(which could simultaneously block contralateral homologs) (Butt and Kiehn 2003). These possible

connections are shown in the schematic in Fig. 3.

Fig. 3 Schematic organization of the network ensuring flexor-extensor alternation during fictive locomotion. Excit-

atory interneurons are shown in red, while inhibitory interneurons are shown in blue. Only one side of the spinal cord isshown. See text for details (From Kiehn 2011)



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Computational Models of the Rodent Locomotor CPGWhile experimental data have generated many schematics of how the rodent locomotor CPGmightbe organized, there are few fully realized computational models that allow testing of hypotheses.Sherwood et al. (2011) presented an abstract model of the rodent locomotor CPG, based oninteractions between coupled oscillators following the unit burst generator (UBG) approach ofGrillner (1981). This model is described in detail in the entry by Sherwood in this volume, so willbe only superficially described here. This model explores the consequences of variable levels andpatterns of excitatory and inhibitory connections between intrinsically oscillatory neurons, rangingfrom a half-center of two neurons with mutual excitation or inhibition to a 12-neuron network withbilateral flexor and extensor oscillators, commissural interneurons, and motoneurons. The neuronmodels are adapted from a conductance-based model of the respiratory network in the pre-Botzinger complex (Butera et al. 1999); oscillatory neurons have an appropriate balance ofpersistent sodium currents (INaP) and leak currents and show spike frequency adaptation. Thefull 12-neuron model required significant tuning of synaptic weights and cellular properties togenerate stable alternation. A more detailed analysis was made of smaller 2- and 4-neuronnetworks coupled with variable excitatory and inhibitory synapses. Of the 4-cell models, mostrapid convergence to the locomotor pattern was obtained with strong inhibition between ipsilateralflexors and extensors, and different levels of inhibition between flexors (and extensors) onopposing sides of the cord, along with moderately strong excitatory connections between contra-lateral extensors and flexors; however, the strength of the synapses had to be very high forsignificant convergence to the locomotor pattern. This model showed the strong dependence ofthe locomotor output on the ratios of strengths of synaptic connections between the components ofthe CPG network.

Rybak and colleagues explored a different set of models of the locomotor CPG, based onexperimental studies of spontaneous deletions in locomotor activity during fictive locomotion.First studied in turtles (Stein 2008) and cats (Duysens 1977; Lafreniere-Roula and McCrea 2005;Duysens 2006) and more recently in mice (Zhong et al. 2012), deletions occur when one or moremotor roots remain silent when they should fire a rhythmic burst of action potentials; for example,the flexor nerves innervating a limb may fall silent while the extensor nerves fire tonically. In cats,about two-third of these deletions resume activity an integer number of locomotor cycles later andare named “non-resetting” deletions; in neonatal mice, almost all of the deletions are non-resetting.This implies that a “clock” continues to tick during the deletion, when there is sometimes norhythmic motoneuron output at all. The remaining deletions reset the phase of the next cycle,presumably through resetting the “clock.”

Rybak et al. (Rybak et al. 2006; McCrea and Rybak 2007) formulated a two-layer model of thecat locomotor CPG that could explain the origin of resetting and non-resetting deletions by positingtwo functional levels in the CPG: a half-center rhythm generator (RG), performing a “clock”function, and determining the rhythmic output of the system, and pattern formation (PF) networksthat are driven by rhythmic input from the RG and coordinate the phasing and intensity of thedifferent motoneuron groups to drive locomotion. This model is described in detail by Shevstova inthis volume, so is only briefly described here. In this model, the RG is symmetrical, with a half-center organization of mutually inhibitory rhythmogenic flexor and extensor modules: these driverespective interneurons in the flexor and extensor PF networks (which are also mutually inhibitory)which in turn drive their motoneurons to generate the behavior. Resetting deletions, which do notresume an integer number of cycles later, arise from errors in the RG network, while non-resettingdeletions, which do resume an integer number of cycles later, arise from errors in the PF network.



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Only one limb was modeled, as the cat electrophysiological data were obtained only from one sideof the animal.

Zhong et al. (2012) carried out a parallel physiological analysis of spontaneous locomotordeletions during transmitter-evoked fictive locomotion in the isolated neonatal mouse spinalcord. This preparation allowed simultaneous recordings from flexor and extensor ventral rootson both sides of the cord. The general outlines of the cat results were affirmed, but with somesignificant differences. First, nearly all (over 90 %) of deletions were non-resetting, suggesting thestability of the RG network in this preparation. Second, the consequences of flexor and extensordeletions were quite different, suggesting a basic asymmetry in the CPG network organization.During a flexor ventral root deletion, extensor motoneurons fired continuously throughout theperiod that the flexors were silent. In contrast, during an extensor ventral root deletion, the flexormotoneurons continued to fire rhythmically at the same cycle frequency seen before the deletion.This suggests that the CPG has an asymmetric flexor-dominated architecture, as suggested initiallyby Pearson and Duysens (1976). Third, virtually all of the non-resetting deletions affected only oneside of the cord: flexor and extensor bursts on the opposite side continued with no disturbance. Suchuninterrupted contralateral bursting is not required for continuation of the ipsilateral rhythm duringnon-resetting deletions, however: non-resetting deletions could be observed with an isolated left orright hemicord, and even from a single isolated hemisegment.

Fig. 4 Two-layer computational model of the rodent CPG for one hind limb. The RG rhythm generator layer is shown

in yellow, while the PF pattern formation layer is shown in blue. Arrows show excitatory synapses, while circles showinhibitory synapses. See text for details (Modified from Zhong et al. 2012)



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The isolated neonatal mouse spinal cord also allowed recordings from identified spinal neuronsfrom the L2 segment during non-resetting flexor deletions (Zhong et al. 2012). Ipsilateral moto-neurons lost their rhythmic excitatory synaptic drive and fell silent during a flexor deletion; itappeared that neurons upstream of the motoneurons were falling silent during the deletion.Commissural interneurons, in contrast, continued to receive rhythmic synaptic drive and firedrhythmically during an ipsilateral motor deletion. The ipsilaterally projecting V2a interneurons fellinto two distinct classes during ipsilateral flexor deletions. Type I V2a interneurons continued toreceive rhythmic excitatory synaptic drive and fired rhythmically during the deletions. In contrast,the type II V2a interneurons acted like motoneurons: these V2a interneurons lost their synapticdrive and fell silent during the ipsilateral flexor deletion. The strength of synaptic drive to this classwas significantly correlated with the strength of the motoneuron bursts. Zhong et al. (2012)hypothesize that the type I V2a INs synapse onto CINs (Fig. 2) and drive CIN activity, especiallyat higher frequencies; these neurons appear to continue to receive RG-related synaptic drive duringnon-resetting deletions and may thus be included in the RG network. The type II V2a INs appear tobe outside the RG network as they lose rhythmic synaptic drive during non-resetting deletions;these may be output neurons that synapse on and help to drive motoneuron activity.

Rybak and colleagues revised their earlier model to incorporate these new data to generatea model of the rodent CPG (Zhong et al. 2012). In this model, the neurons are modeled by Hodgkin-Huxley-style differential equations for the soma and the dendrite compartments, with differentdistributions of active currents and their parameters in each compartment, following the methods ofBooth et al. (1997). Synaptic interactions are modeled by a time-dependent conductance and thedriving force. Each type of neuron is represented by a population of neurons (between 50 and 200for each type), with variable initial parameters and synaptic strengths.

Fig. 5 Two-layer bilateral model of the rodent CPG for locomotion. The RM rhythm generator layer is shown in

yellow, the PF pattern formation layer is shown in blue, and the commissural network is in pink. Arrows show

excitatory synapses, while circles show inhibitory synapses. See text for details (Modified from Zhong et al. 2012)



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Initially, Rybak and colleagues modeled the CPG on only one side of the cord (Fig. 4). As withthe cat model, this model has two layers, a rhythmogenic RG layer and an organizing PF layer.However, the organization of the RG layer is asymmetrical, to explain the asymmetric conse-quences of flexor versus extensor deletions in the mouse spinal cord. In the RG layer, only theflexor group (RG-F) neurons show intrinsic rhythmic activity, produced by an INaP-dependentmechanism with mutual synaptic excitation among members of the group. The extensor group(RG-E) does not have sufficiently large INaP to be endogenously rhythmic and fires tonically. Thesegroups drive major populations in the PF network (PF-F and PF-E) which drive locomotor activityin flexor and extensor motoneuron populations, respectively. These reciprocally inhibit one anotherthrough inhibitory interneurons. The PF-F group receives rhythmic drive from the rhythmogenicRG-F pacemakers, while the PF-E group is tonically excited by the RG-E group and firesrhythmically due to rhythmic inhibition from the PF-F group. This model can produce robustlocomotor-like activity at each level of the network.

The model was then extended to include both sides of the spinal cord, with left and right RGnetworks connected by inhibitory commissural interneuron pathways between the flexor RG half-centers, excitatory commissural pathways between flexor centers, and contralateral extensorcenters, as well as weaker flexor-flexor and extensor-extensor pathways that could maintainsynchrony in the absence of inhibition (Fig. 5). Exploration of this model showed that it couldreproduce the experimental deletion results (Zhong et al. 2012). For example, hemisection of thecord (modeled by elimination of commissural pathways) slows the cycle frequency by half whilemaintaining flexor-extensor phasing; analysis showed that the slowing arose primarily from loss ofexcitatory drive from the contralateral RG-E populations to the rhythmogenic ipsilateral RG-Fpopulations. The model could reproduce the asymmetrical effects of deletions described above. Forexample, non-resetting flexor deletions resulted in ipsilateral tonic extensor activity but unchangedcontralateral activity; this could be obtained by simulated suppression of excitatory input from theipsilateral RG-F population to the ipsilateral PF-F population; this relieved the PF-E populationfrom rhythmic inhibition to allow it to tonically drive the extensor motoneurons. Simulated loss oftonic input from the nonrhythmic RG-E population silenced the PF-E and extensor motoneuron

populations with no effect on rhythmic drive from the RG-F population to the PF-F and flexormotoneurons. These deletion results could also be obtained by loss of synaptic drive at other pointsin the network. Resetting deletions, which are rare in mice, could be simulated by perturbations atthe RG level, for example, temporary inhibition of the rhythmic RG-F population; this is predictedto affect the contralateral population as well.

Preliminary attempts have been made to place the identified interneuron classes within theformal organization of the asymmetric locomotor CPG model. Type I V2a interneurons, whichcontinue to oscillate during non-resetting deletions, are modeled to act within the RG network,though since they do not have rhythmogenic properties they are probably driven by other neuronalpopulations. Type II V2a interneurons, which lose synaptic drive and fall silent during non-resetting deletions, are modeled to be among the output paths from the PF network to motoneurons,though they are not the only population of neurons to do so. As described above, many of the otherdefined interneuron classes are heterogeneous and will require additional molecular analysis tosubdivide them into single classes. However, among the neurons that could act within the RGnetwork are the endogenously rhythmic subset of dI6 interneurons and a subset of Hb9 interneu-rons. Among outputs to motoneurons are another subset of Hb9 interneurons. V1 and V2binterneurons form the Renshaw cells and Ia inhibitory INs in the output layer from the PF network;they could also form the inhibitory interneurons that are modeled in the PF and RG networks.Finally the commissural pathways are composed of inhibitory V0 and excitatory V3 interneurons.



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The model provides predictions of the patterns of synaptic drive that interneurons at differentpoints in the network will receive, both under normal conditions and during deletions of variouskinds. Much further work will need to be done to measure these synaptic interactions and verify orcorrect these predictions.

Future work on models of the rodent spinal locomotor CPG will be able to generate hypothesesfor the roles of the various subsets of the cardinal identified interneuron classes and of their synapticinteractions. At present, our knowledge of the interneurons, and the existence and properties ofsynapses between them, is fragmentary but is the focus of much concerted experimental effort. Theinteraction of modeling with experimental work should rapidly push forward our understanding ofthe rodent locomotor CPG, which can serve as a model for other, more complex neural networks inthe nervous system.

Cross-References

▶Computational Analysis of Rodent Spinal CPG▶Two-Level Model of Mammalian Locomotor CPG

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Locomotor Pattern Generation in the Rodent Spinal …pages.nbb.cornell.edu/neurobio/harris-warrick/148.pdf · Locomotor Pattern Generation in the Rodent Spinal Cord Ronald Harris-Warrick*