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Review

Origin of excitatory drive to a spinal locomotor network

Alan Robertsa,⁎, W.-C. Lia, S.R. Soffea, Ervin Wolfb

aSchool of Biological Sciences, University of Bristol, Woodland Road, Bristol, BS8 1UG, UKbDepartment of Anatomy, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary

A R T I C L E I N F O

⁎ Corresponding author. Fax: +44 117 9257374E-mail address: a.roberts@bristol.ac.uk (A

0165-0173/$ – see front matter © 2007 Elsevidoi:10.1016/j.brainresrev.2007.06.015

A B S T R A C T

Article history:Accepted 17 June 2007Available online 27 July 2007

A long-standing hypotheses is that locomotion is turned on by descending excitatorysynaptic drive. In young frog tadpoles, we show that prolonged swimming in response to abrief stimulus can be generated by a small region of caudal hindbrain and rostral spinal cord.Whole-cell patch recordings in this region identify hindbrain neurons that excite spinalneurons to drive swimming. Some of these hindbrain reticulospinal neurons excite eachother.We consider how feedback excitationwithin the hindbrainmay provide amechanismto drive spinal locomotor networks.

© 2007 Elsevier B.V. All rights reserved.

Keywords:FeedbackLocomotionReticulospinalRhythm generationSynapsesXenopus

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221.1. Xenopus tadpole swimming and a minimal circuit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231.2. Excitatory neurons that drive swimming. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231.3. Excitatory connection between hindbrain neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

2. Howmight networks with feedback excitation function? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

1. Introduction

In many animals, a simple stimulus can initiate prolongedepisodes of locomotion. Alternatively, locomotion can startspontaneously as a result of something happening inside thenervous system. In both cases, it is assumed that networks ofpremotor neurons are able to generate a basic pattern of motor

.. Roberts).

er B.V. All rights reserved

output to drive rhythmic locomotor movements provided theyreceive suitable excitatory drive. So, in experiments, artificialdrive is often provided by applying chemical excitants (e.g., inlamprey, crayfish and mammal; Buchanan and Cohen, 1982;Mulloney, 1997; Nakayama et al., 2004). This drive is usuallyassumed to come from higher nervous centres (e.g., insects;Ludwar et al., 2005), and in vertebrates from reticulospinal

.

Fig. 2 – The tadpole and swimming activity. (A) HatchlingXenopus tadpole with CNS and segmented swimmingmuscles. (B) Motor nerve recording (vr) with alternatingactivity. (C) frequency plot of swimming initiated by a 0.5-mscurrent pulse in a tadpole with most of the brain removed.Plot shows prolonged activity which stops spontaneously (*).

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neurons in the brainstem (Garcia-Rill and Skinner, 1987;Matsuyama et al., 2004; Noga et al., 2003).

The idea that locomotion occurs as a result of descendingexcitatory drive came from classic experiments done by GrahamBrownondecerebratecats supportedso theirhindlimbswere freetomove (Brown, 1911, 1914). After cutting through all the sensoryroots in the lumbar region to abolish any reflexes involving thehindlimbs, Brown cut through the whole spinal cord. This briefstimulus led to rhythmic alternatingmovements of thehind legs.Brown concluded that these movements were generated insidethe spinal cord without reflexes. He proposed that his lesion tothe spinal cord led to a steady, injury dischargewhich in turn ledto tonic excitation ofwhat he called “half-centres” controlling themuscles at each joint of the leg (Fig. 1). As a result, onehalf-centrebecame active and inhibited the other. He also proposed that thisreciprocal inhibition fatigued quickly so the second half-centrecould become active after a delay. In this hypothesis, rhythmgenerationdependson tonic excitation, reciprocal inhibition, anda fatigue process that introduces a delay and effectively sets thecycle period for the rhythm.A cornerstone of Brown's hypothesisis descending tonic excitation providing the synaptic drive toactivate rhythmic locomotor movements.

If tonic excitation drives locomotion, theobvious question is:where does it originate? Work on mammals showed thatlocomotion can be activated during stimulation of so-called“locomotor regions” in the brain (Garcia-Rill and Skinner, 1987;Noga et al., 1988, 2003; Shik et al., 1966). These regions arethought to activate reticulospinal neurons that then excite thespinal networks (Brocard and Dubuc, 2003; Ohta and Grillner,1989; Orlovsky, 1970a,b; Viana Di Prisco et al., 1997, 2000). So,pathways have been identified but the origin of the activity inthese pathways and the mechanisms for generation of contin-uous activity to drive locomotion remain unclear.

To investigate where the basic drive to activate spinalcircuits during locomotion arises and what keeps it going aftera brief stimulus initiates locomotion, we have studied a simplecase, the hatchling frog tadpole. Recentwhole-cell recordings inthe tadpole hindbrain have revealed neurons that may be thesource of the excitatory drive that turns on and sustainsswimming locomotion (Li et al., 2006).

1.1. Xenopus tadpole swimming and a minimal circuit

When it hatches from the egg at an age of 2 days, the Xenopustadpole is 5 mm long (Fig. 2A) and will swim rapidly when it is

Fig. 1 – Brown'shypothesis. In thespinal cord, theantagonistic“half-centres” containing the motoneurons (mn) for flexorsand extensors of each joint are connected by reciprocalinhibition (closed circles). Alternating activity is present so longas there is descending tonic excitatorydrive to thehalf-centres.

touchedon the tail skin. If the tadpole is immobilised inα-bungarotoxin to block the neuromuscular synapses, recordings can bemade from the motor nerves or ventral roots innervating thetrunk swimming muscles. Just as in the moving tadpole, whenthe skin is touchedor a 1mselectrical pulse is given toexcite thesensory nerve branches in the skin, alternating bursts of activityare seen in the ventral roots. This is fictive swimming (Fig. 2B;Kahn and Roberts, 1982). As in the swimming tadpole, fictiveswimming can last formany seconds or evenminutes followinga brief stimulus. It usually starts fast, slows down gradually andfinally stops spontaneously (Fig. 2C).

To locate the neurons required for prolonged swimmingactivity we used lesions (Li et al., 2006). If the brainwas removedrostral to the seventh rhombomere (just caudal to the vagus),fictive swimming activity lasted as long as in the intact animal(Fig2C). In spinal tadpoles, swimmingonly lasted fora fewcycles.However, a 0.4-mm-long region of the caudal hindbrain androstral spinal cord produced swimming episodes that were notsignificantly shorter than preparations where only the brainrostral to the seventh rhombomere was removed. These exper-iments showed thatneuronsandnetworks sufficient to generateprolonged swimming lay in a very small region of the CNS.

1.2. Excitatory neurons that drive swimming

Our objective was to find the neurons in the caudal hindbrainthat produced excitation to drive swimming. Earlier recordingsanddye fillings of spinalneuronshadshownthatneuronswithdescending axonal projections were active during swimmingand released glutamate to excite spinal neurons by activatingAMPA and NMDA receptors (Dale and Roberts, 1985). Theseneuronswere called descending interneurons (dINs). Anatom-ical and lesion studies suggested that they formed a column ofrelatively ventral neurons extending from the rostral spinal

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cord into the hindbrain (Roberts and Alford, 1986). We madewhole-cell patch recordings from pairs of neurons in fairlyventral positions in the caudal hindbrain and rostral spinalcord thatwereactiveduring swimming (Li et al., 2006).Neuronswere found which fired earlier than other neurons on eachcycle of swimming and produced excitation in more caudalspinal neurons (Fig. 3). The neurons thatwere excited includedall types that are normally active during swimming includingmotoneurons, reciprocal inhibitory interneurons, recurrentinhibitory interneurons and other excitatory interneurons (seeRoberts, 2000). We call these excitatory hindbrain neuronshindbrain descending interneurons (hdINs).

Examining the pharmacology of the excitation produced bythese neurons led to a major surprise. The EPSPs they producewerenot blockedcompletely by glutamate receptor antagonists.It turned out that these neurons corelease glutamate and ace-tylcholine at this stage of the tadpole's development (Li et al.,2004b).Their excitation isonly fullyblockedby theapplicationofAMPAR, NMDAR and nAChR antagonists together. At present,the functional significance of this corelease is not understood.

Fig. 3 – Activity, synaptic connections and properties of excitatopositions of hdIN and spinal commissural IN (cIN). (B) hdIN and cstimulus at arrowhead. (C) Grey section in B expanded to show hthan cIN spikes. (D) Current induced action potential in hdIN leadevokes only a single action potential. (F) during depolarisation, n

The excitatory neurons had some other uncommon char-acteristics.When their action potentialswere compared to thoseof all the other neurons active during swimming they weresignificantly longer in duration (Fig. 3C). When long currentpulseswere injected, theseneurons firedasingleactionpotentialat the start of the pulse whereas other spinal interneurons firedrepetitively (Fig 3E; Aiken et al., 2003; Li et al., 2002, 2004a). At theend of depolarising current pulses there was never any evidencefor prolonged plateau potentials (Kiehn, 1991). Finally, theexcitatory neurons showed post-inhibitory rebound but only ifnegative current pulses were injected while the neurons weredepolarised from their resting membrane potential (Fig. 3F).These properties will become significant when we discuss thepossiblemechanisms for the generation of prolonged responses.

When we looked at the anatomy of the excitatory neuronsrecorded in the caudal hindbrain,we found thatmore than50%of them had an ascending as well as a descending axon. Thisraised the intriguing possibility that the group of excitatoryneurons in the hindbrain that were active during swimmingcould excite each other.

ry hindbrain neurons (hdIN). (A) Diagram of the CNS withIN fire ones pike per cycle during swimming initiated by skindIN spikes are longer in duration and earlier in the cycles to excitation of cIN at latency of 1.5 ms. (E) Positive currentegative current pulses can lead to firing on rebound.

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1.3. Excitatory connection between hindbrain neurons

The excitatory neurons that we have described lie in the ven-trolateral, caudal hindbrainandhavedescending axonsproject-ing into the spinal cord. These neurons are the reticulospinalneurons of early development (van Mier and ten Donkelaar,1989). If their activity can drive spinal neurons during swim-ming, a major question is how their own activity is prolongedfollowing a brief stimulus. Since they do not show any signs ofcellular-based bistability or prolonged plateau potentials, theother main hypothesis is that their activity results from somekind of network interaction. The most obvious of these ismutual excitation that could be the basis for network reverber-ation (Hebb, 1949; Lorente de No, 1938). Since some hdINs haveascending and descending axons, we therefore looked forevidence of mutual excitatory interconnections between them(Li et al., 2006).

In a sample of whole-cell recordings from 47 pairs of hdINs,nearly 30% of the caudal neurons in the pair had an ascendingaxon and, in these cases, current-evoked action potentials ineither neuron led to excitation of the other. An example of thisreciprocal excitation is shown in Fig. 4. The excitation led tolarge EPSPs at short and constant latencies which were onlyblocked by the joint application of glutamate and acetylcho-line antagonists. These synaptic connections were thereforedirect and monosynaptic.

The significance of these neurons and their reciprocalexcitation was emphasised in preparations bathed in 0 Mg2+

Fig. 4 – Reciprocal excitation between hindbrain neurons. (A) Insshown in lateral view.Bothhavedescending (d) axons.Thedescencontacts at arrows. (B) Intracellular current injection (at arrowhealarge EPSP at short and constant latency. (C) Diagram summariserepresent excitatory synapses).

saline so the NMDA component of glutamate EPSPs could beseen. These preparations are more excitable and in a numberof cases stimulation of one hdIN led to a feedback EPSP fol-lowing the action potential. In a few cases, stimulation of onehdIN activated swimming in the whole circuit.

The critical role of the caudal hindbrain where the hdINs lieand feedback excitation among them activating NMDA recep-tors was endorsed by experiments on tadpoles with the brainremoved rostral to the 7th rhombomere. A pulse to the skinwillinitiate prolonged swimming but if theNMDARantagonist D-AP5 is perfused onto the caudal hindbrain 4 s after swimmingstarts, the swimming slows down and stop prematurely.

2. How might networks with feedbackexcitation function?

We now need to consider how a group of hindbrain reticulosp-inal neurons with some rather unusual properties and recipro-cal excitatory connections could allowanetworkof neurons inasmall regionof the tadpole's hindbrain and rostral spinal cord toproduce prolonged swimming activity. Fortunately the tadpo-le's nervous system is simple enough that the main spinalneuron classes active during swimming have been charac-terised (Li et al., 2002, 2004c, 2006; Roberts, 2000). Furthermore, ahypothesis has been put forward to explain prolonged rhythmicswimming activity following a brief skin stimulus (Dale andRoberts, 1985; Roberts and Tunstall, 1990).

et shows location of two hdINs, with detailed morphologyding axonofhdIN2branches (*) to ascend (*). Possible synapticds) shows that each hdIN excites the other producing as anatomy and interactions between hdIN pair (triangles

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The hypothesis for tadpole swimming is based on a simplenetworkofneuronsand connectionswith strong similarities toBrown's half-centre proposals (Fig. 1). We have made aphysiologically realistic model of this network using simple,single-compartment model neurons with the cellular proper-ties of the excitatory interneurons (dINs and hdINs; Li et al.,2006).Wecanuse thismodel to explain and test theplausibilityof our hypothesis (see also Roberts and Tunstall, 1990). Ourbasicmodel network is shown in Fig. 5A. The antagonistic half-centres on the left and right sides of the nervous system con-tain populations of neurons forming longitudinal columns.Each half-centre is excited by a single EPSP from the sensorypathway given to the right side 20 ms after the left side. Therearemotoneurons (mns)whichwould normally activate the leftand right trunk swimming muscles, glycinergic inhibitoryinterneurons (iINs) that inhibit all neuronson the opposite side(reciprocal inhibition), and excitatory interneurons (hdINs)that produce fast AMPAR/nAChR and slow NMDAR-mediatedexcitation of all neurons on the same side.

The basic model operates as follows. When the sensoryexcitation reaches the left side, all neurons fire a single action

Fig. 5 – Networks producing prolonged swimming. (A) Themodel half-centre network of the tadpole spinal cord wherelarge circles arepopulationsofneurons, triangles are excitatorysynapses, small circles are inhibitory synapses, synapses go toall the neurons in a half-centre. (More details in text.) (B) hdINswith only descending axons for feedfoward excitation. (C)hdINswith ascending and descending axons to allow feedbackexcitation. (D) Number of neurons active in the populationmodel following sensory excitation at time 0.

potential. The hdINs feedmixedAMPARandNMDARmediatedlong-duration excitation back to all left-side neurons. They aretherefore depolarised but do not fire again because they arerather inexcitable when depolarised. Left-side firing leads toglycinergic inhibition of all right-side neurons just before theyreceive their sensory excitation. All the right-side neuronsthen fire as the IPSPdecays. Feedback excitationnowgoes to allright-side neurons but they are depolarised so they do not fire.The right side sends inhibition back to the left side. This occursduring the long-feedback EPSP, hyperpolarises the neurons,allows their excitability to recover and they fire on rebound asthe IPSP decays. Activity will now reverberate from side to sideindefinitely, producing a pattern of alternation just like thatseen in neurons during swimming (Fig. 3B).

Unlike the real nervous system, our basic model has nolength. We therefore added a length dimension to make apopulation model of the short 0.4-mm-long piece of hindbrainand spinal cord that can generate prolonged swimming (seeSection 1.1). Anatomical studies have shown that theremay beabout 8 neurons of each class on each side per 0.1mm(Yoshidaet al., 1998) so we used 90 neurons on each side. The reciprocalinhibitory neuron (iIN) axons branched to ascend and descendandwere long enough to contact all neurons on the other side.To test the significance of the axonal projection patterns of theexcitatory neurons (hdINs), we made two model networks. Onone hand, the hdINs had only descending axons (Fig. 5B) andon the other, they had ascending axons as well (Fig. 5C). Boththese networks could generate swimming activity followingsensory excitation, but without the ascending axons theactivity failed after only a few cycles (Fig. 5D). On the otherhand, with ascending axons, which allow the hdINs to exciteeach other as a group, the activity continued indefinitely.

3. Conclusions

It is no surprise that the excitation to drive swimming in thehatchling frog tadpole comes from hindbrain reticulospinalneurons releasing glutamate and that one of the mechanismsinvolved is reverberation based on feedback excitation. Howev-er, a number of features of the hindbrain excitatory reticulosp-inal neurons are rather unexpected:

(a) These neurons corelease glutamate and acetylcholine.Thismay be a transitory developmental feature but, as inmotoneurons in developing mammals (Nishimaru et al.,2005), its functional significance is not yet clear.

(b) The reticulospinal neurons in the hindbrain appear toform a continuous longitudinal column of neurons withspinal excitatory descending interneurons. All theseneurons share similar membrane properties, like firing asingle action potential to current, and all corelease gluta-mate and acetylcholine. The ascending axons which areonly present in thehindbrainneurons allow them to forma group with mutual, feedback excitation, whereas thespinal neurons with only descending axons can onlyproduce feedforward excitation.

(c) The hindbrain reticulospinal neurons that drive swim-ming fire in the same pattern as motoneurons. They arecomponent parts of a central pattern generator that

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extends into the spinal cord and provides feedbackexcitation to sustain its ownactivity. This contrastswithBrown's original proposal where the drive was steadybut the activity it producedwas alternating bursts. It alsocontrasts with most other vertebrates where the neu-rons producing drive are assumed to form separate sys-tems from those generating locomotor rhythms.

(d) It is critically important that the feedback EPSPs tohindbrain neurons are NMDARmediatedwith durationswhich are long compared to the swimming cycle period.This allows them to sum from cycle to cycle (Dale andRoberts, 1985) so the neurons can fire on rebound afterthe short-duration reciprocal inhibition from the oppo-site side.

(e) It is also critical that the feedbackexcitation to excitatoryneurons is controlled primarily by their own inability tofire repetitively but also by recurrent synaptic inhibition(Li et al., 2004c).

It will be interesting to see whether features of the networksand mechanisms we have described for these hindbrainreticulospinal neurons driving locomotion in a very small frogtadpole will also be found in larger adult animals moving atlower frequencies.What is clear is that we have provided directevidence for excitatory synaptic feedback between neuronsactive in a network able to produce prolonged responses to briefstimuli. This is at last concrete evidence for a very well estab-lished hypothesis.

Acknowledgments

WethanktheWellcomeTrust andHungarianNationalResearchFund OTKA T037522 for supporting our research.

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