Neural Mechanisms Generating Locomotion in Mammals

8/12/2019 Neural Mechanisms Generating Locomotion in Mammals

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Neural m echanism s generating locomotion studiedin mammalian brain stem -sp inal cord in vitro

JEFFREY C. SM ITH,*t JACK L. FELDMAN,*t AND BRIAN J. SCHMIDT* l*Systas Neurobiology Laboratory, Department of Kinesiology, U niversity of California, Los Angeles, Los Angeles,Ca4fornia 90024, USA , and Department of Physiology, Northwestern University, Chicago, Illinois 60611, USA

ABSTRACTT he n eu ra l c on tr ol s ys te m f or g en er at io n o f l oc om ot io nis an im portant system for analysis of neural m echanism sunderly ing complex m otor acts. In these studies, a novelexperim ental model using neonatal rat brain stem andspinal cord in vitro was developed for investigation ofthe locomotor system in mammals. The in vitro brainstem and spinal cord system was shown to retain func-tional circuitry for locomotor command generation ,m otor pattern generation, and sensorim otor integration .T his system w as exploited to investigate neurochem icalmechanisms involved in neurogenesis of locom otion.Evidence was obtained for peptidergic and y-am ino-butyric acid-m ediated m echanism s in brain -stem cir-cuits generating locomotor commands. Chobinergic,dopaminergic, and excita tory am ino acid-mediatedmechanisms were shown to activate spinal cord circuitsfor locomotor pattern generation . Endogenous N-methyb-D-asparticacid receptorsin spinal netw orks werefound to play a central role in the generation of loco-motion. The chemically induced patterns of motor activ-ity and rhythm ic membrane potential oscillations ofspinal motoneurons were characteristic of those duringlocomotion in other mammals in vivo. The in vitro brainstem and spinal cord model provides a versatile andpowerful experim ental system with potentially broadapplication for investigation of diverse aspects of theneurobiology of mammalian motor control system s.-SMITH,J. C.; FELDMAN,J . L.; SCHMIDT , B.J. Neuralm echanism s generating l oc om ot io n s tu di ed i n m am -malian brain stem -sp inal cord in vitro . FASEBJ. 2:2283-2288; 1988.Key W ord,: neural pattern generation m otor control systemsex cita to ry a min o a cid n eu ro tr an sm itte rs N -m eth yl-D -a sp ar tica cid r ec ep to rs

and network properties underly ing the generation oflocomotion and other rhythm ic movements of brain stemand spinal cord origin is a necessary step toward under-standing central nervous system (CNS) control of com -plex motor acts (3, 4). Identifying neuronal membraneand neurochem ical mechanisms responsible for the sig-nal processing and interneuronal communication thatunderlie these acts is fundamental. These mechanism shave not been identified (2), largely because of thedifficulty in perform ing neurochemical and membranebiophysical studies in the mammalian CNS in vivo. Asa step toward overcom ing these lim itations, we haverecently developed in vitro preparations of the mam -malian brain stem and spinal cord that provide a novelexperim ental system for investigation of motor controlsystem s for rhythm ic movements (5-8; see also ref 9). Thein vitro system offers several important advantages overconventional in vivo approaches, including precise controlof CNS extraceblular environment and mechanical sta-bility for bong-term intracellular recording, that arenecessary for detailed analysis of neural mechanism s.

In the work reported here, w e have extended ourearlier observations on viability of locomotor and othermotor circuits in the in vitro brain stem -spinal cord(5-8) and have now established that th is system retainsfunctional circuitry for the major neural components ofthe locomotor control system , including circuits formotor command generaton, motor pattern generation,and sensorimotor integration . W e exploited this systemto identify endogenous neurochem ical mechanisms in-volved in motor command and pattern generation . Ourdata indicate that diverse neurochem icab mechanism sare involved, and that there is an essentia l role for ex-citatory am ino acids acting at N -methyl-D -asparticacid (NMDA) receptors in locomotor pattern genera-tion . S im ilar findings have been obtained in bower ver-tebrates (3, 10-12), and our results w ith the mammaliansystem are consistent w ith the hypothesis (5, 13) thatthere is an evolutionary conservation of mechanism s forrhythm ic locomotor pattern generation in vertebrates.

THE NEUROGENESIS OF LOCOMOTION in vo lv es in te ra ctio nof brain-stem networks for generation of motor com -mands, spinal cord motor pattern-generating networks,and sensorimotor circuits (1-4). Analysis of neuronal O n leavefrom LaboratoryofN eural Control, N IN CD S, N ationalI ns ti tu te sf H ea lt h, B et he sd a, M D 2 08 92 .


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RESULTSIsolated brain stem and spinal cord from 0- to 4-day-oldSprague-Dawley rats were maintained under in vitroconditions2 in an experim ental recording chamber aspreviously described (7). The neonatal ra t CNS ha sseveral properties that perm it viability for extendedperiods in vitro .3 Brain-stem or spinal locomotor circuitrycould be repeatedly activated by selectively applyingspecific neurochem icals to the brain-stem or spinal cordbathing solution in preparations where the in vitro bathwas partitioned at the spinomedullary junction with abarrier (7).2 S tereotypical patterns of rhythmic loco-motor activity in spinal ventral roots and individualmotoneurons (Fig. 1) could be generated for periods ofup to 8 h. The locomotor nature of the motor patternswas verified with preparations retain ing pairs of inner-vated hindbimbs or forelimbs in vitro2 (6-8; see also ref16). The chem ically evoked motoneuronal activity pro-duced mubtijo in t, alternating steppinglike movements ofthe limbs. E lectromyographic (EMG) recordings fromhindbimb muscle confirm ed the presence of alternatingpatterns of flexor-extensor muscle activation charac-teristic of those during locomotor movements in intactquadrupeds. The patterns of activ ity of flexor-extensormuscles acting at the hip (sartorius, sem imembranosus;Fig. 1B , knee (sem itendinosus, rectus femorus), andankle (tibialis anterior, gastrocnem ius; see also ref 16)were sim ilar to those described during quadruped walk-ing (17). The patterns of limb motor nerve activityrecorded extracellularby, as well as the rhythm ic oscilla-tions of motoneuron membrane potential recorded in-traceblularby (Fig. 1C), were also c ha ra ct er is ti c o fquadruped walking (1, 14, 17). Patterns for other loco-motor gaits such as galloping could be generated byneurochem ical activation of brain-stem circuitry.

The chem ically induced motor patterns resembledthose elic ited by activating spinal sensory afferent path-ways. P inching the tail or skin at the base of the tail inpreparations retain ing hindlimbs and tail, or directelectrical stimulation of lumbar dorsal roots, elicitedtransient bouts of motoneuronal activ ity for walking(Fig. 2). The central pattern-generating mechanism re-mained responsive to peripheral afferent inputs forperiods of up to 7-8 h, indicating long-term viability ofsensorimotor reflex pathways. The correspondence be-tw een the motor patterns evoked chem ically and byphysiological stimuli (e.g., ta il p inch) that induce loco-motion in vivo (1) suggests that the neurochem icals usedto elicit locomotor output activated normal patterngeneration m echanism s.

S tim ulation of several locom otor com mand-generatingregions in the brain stem in vivo can activate spinallocomotor networks via descending bulbospinal motorcommand pathways (1, 19, 20). W e specifically testedneurochem icals likely to activate the mesencephaliclocomotor region (M LR), a major command-generatingarea in the m idbrain tegmentum receiving input fromthe motor cortex and basal ganglia (20). W ith thebrain stem transected in vitro at the m idcollicular levelto retain the MLR, the excitatory am ino acid L-

glutamic acid, the neuropeptide substance P, or antag-onists to -y-am inobutyric acid (GABA) (bicuculline ,picrotoxin) applied to the solution bathing the brainstem induced locomotor activity (Fig. 1, D an d E .Threshold concentrations of GABA antagonists or sub-stance P for induction of locomotor output were 1-10tM . The onset of motor activ ity occurred within 5-10mm after bath application of the neurochem icals. Themotor output induced by substance P frequently sub-sided after 15-30 mm , probably ow ing to inactivationof the peptide in the CNS , whereas the motor activ ityinduced by GABA antagonists continued for severalhours. The motor activ ity typically consisted of recurringepisodes of 10-30 cycles of rhythmic motor dischargewith interlimb coordination patterns for walking (F ig.1D), with initially high discharge frequencies andamplitudes that progressively declined as the bout oflocom otor activity subsided. The episodes of rhythmicactivity were separated by 20- to 60-s periods of low-level tonic motoneuronal activity where presumablyspinal neuron excitation was insuffic ient to activate themotor pattern generation system . L-Gbutam ic acid(0 .5-1 mM) adm inistered with the am ino acid uptakeinhibitor dihydrokainic acid (DHK ; 0.2 mM) producedeither transient locomotor activity n = 4), or recurringepisodes n = 5) sim ilar to those induced by GABA an-tagonists. The average cycle durations recorded duringrhythmic activ ity induced by bicuculline (5-ho tM ) orpicrotoxin (10 j sM) [2.6 0.6 n = 7) and 2.5 0.5 sn = 2) , r es pe ct iv el y] w er e s im il ar to c yc le d ur at io nsafter activation w ith substance P (5 LM ) (3.1 0.6 s,n = 4) or L-glutam ic acid (1 mM) (2.5 0.4 s, n = 5) ;the lowest cycle periods in all cases were 1.2-1 .4 s. Thesecycle periods are at the upper end of the range expectedfo r intact quadrupeds during ambulation (1).

The above findings are consistent w ith studies in vivo(19, 20) that suggest the presence of GABA-mediatedinhibitory synaptic inputs, and substance P -mediatede xc it at or y i np ut s t o t he MLR from basal ganglia outputnuclei. There is evidence (20) that substance P may

2The standard bathing solutioncontained 128 mM N aC I, 3 .0mM K CI , 0 .5 mM NaH2PO4, 1.5 mM CaCl2, 1.0 mM MgSO4, 21mM NaHCO3, 30 mM glucos e,equili bratedith 95 02-5 CO2(pH 7.4) at 27 {176}C.h e i n v it ro b at h w as p ar ti ti on eda t t hespinomedullaryjunctionwith a vaselinearriertoisolatehebathingsolutionsorthebrainstem an d spi nalcor d,w hich allow ed indepen-dent manipulation of the ionic or neurochem ical concen trations inthe brain-stem and sp inal cord so lutions. W ith preparations retain-ing limbs, the skin is rem oved from the limbs and muscle viabilityis m ain tained by the bathing m edium .3The sm all size of the neonatal rat b rain stem and spinal cordw ith short d iffusion distances allows m aintenance of CNS viabilityby diffusion of 02 and m etabolic substrates from the bath ingmedium . The to lerance of mammalian neonatal CNS to hypoxiaand anoxia further perm its m aintenance of neuronal energymetabolism and viab ility (7). V iability of brain stem -sp inal cordcircu its could be m aintained in v itro in some preparations forperiods of up to 8-12 h.

4Evidence suggests that th e MLR corresponds in part to thepedunculopontine nucleus (PPN ) (20). Developm ental studies (21)have verified a well-defined PPN in neonatal rat brain stem .

5The longer locomotor cycle periods in vitro resu lt in part fromthe lower CNS tem perature.


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NEURAL MECHAN ISMS FOR LOCOMOTION STUDIED IN VITRO 2285

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Figure 1. Patterns of motor activ ity generated in v itro by chem ical activation of locomotor circuitry . A Hindlimb and forelimb ventralroo t activ ity elicited by 0.25 mM L-aspartic acid (ASP) w ith 0.2 mM dihydrokam nic acid (DHK) in spinal cord bath (see Fig . 3), illustratinga general pattern of ven tral root activ ity induced by chem ical activation of sp inal circuitry . R ecord ings were obtained w ith suctionelectrodes on left (L ) and right (R ) ventral roo ts at the six th cerv ical (C 5) and fifth lum bar (L5) segm ent levels . Motor ou tpu t on a g ivenventral roo t consists of rhythm ic d ischarge w ith two phases corresponding to lim b flexion and extension . For each trace shown, the ventralroo t discharge correlated main ly w ith flexor muscle activ ity as determ ined by limb electromyogram s (EMG); extensor activity could berecorded (see B on other ventral roo ts. Phase lag of 50 of the cycle period (i.e ., 180 {176}hase difference) occurs betw een ipsi- andcontralateral ventral root flexor (or extensor) activ ity recorded at the same segmental level fo r all cycle frequencies. The phase lag betweenipsilateral hindlim b and forelimb flexor (or extensor) activity was less than 10 of cycle period . These temporal patterns are characteristicof one type of quadruped walk ing (1). M otor patterns for other gaits such as galloping where h ind limb movem ents are synchronous,alternating w ith synchronous forelimb movements, were generated by activation of brain -stem locomotor regions (see E . B Recordingsof hindlim b EMGs during lim b movements induced by activating sp inal circuitry w ith L-aspartic acid and DHK . T races show activityof h ip flexor muscle Lsarto rius (Sart)] and hip extensor muscle [sem im embranosus (Sm )l w ith alternating pattern of flexor-extensoractivation characteristic of quadruped locomotion. C L5 ventral roo t recording and in tracellu lar record ing of activ ity of ipsilateralmotoneuron (nonspik ing) in L5 segm ent show ing rhythm ic locom otor drive potentials (LDPs) in motoneuron (MN) during locomotioninduced by N -methyl-DL-aspartic acid (NMA) applied to sp inal cord . M otoneuron resting membrane potential w as -52 mV . Intra-cellular record ing was obtained w ith a m icroelectrode filled w ith 3 M potassium acetate. LDPs like those recorded in vitro have beendescribed in cat motoneurons (14), as well as in lower vertebrates (15), and are believed to reflect alternating excitato ry and inh ibito rydrive to m otoneurons (14). D, E Examples of h ind limb ventral root discharge patterns w ith activation of brain -stem circuitry by y-am ino-butyric acid (GABA) antagonist b icucu lline (BC) show ing alternating m otor patterns for walking D and synchronous d ischarge patternf or g al lo pi ng E generated in preparations retaining the mesencephalic locom otor reg ion . Average cycle frequency w ith gallop ing patternwas 1.0-0.8 Hz. A continuous sequence of motor activity starting w ith m otor patterns for walk ing fo llowed by patterns for tro tting andthen galloping could be generated by blocking GABAergic inhib ition in some preparations. The pattern of activ ity evoked by GABAan tagonists w as dose-dependent; m otor patterns as shown were typically induced w ith bicuculline concentrations < 20 M . SubstanceP or L -g lutam ic acid induced walk ing patterns sim ilar to those in D.also mediate excitatory connections between the MLRand neurons in the ventromedial medullary reticularformation, a proposed relay for descending transmis-sion of locomotor commands. Consistent w ith th is hy-pothesis, substance P also induced motor patterns forwalking in vitro with the brainstem transected at thepontomedullary junction, excluding the MLR, althoughlocomotor cycle durations (8.9 2.6 s w ith 10 tM sub-stance P , n = 4) were typically longer than in prepara-tions retaining the MLR. Locomotor command gener-ation at the supraspinal bevel probably involves acomplex system of excitatory and inhibitory connectionsamong the basal ganglia, MLR, and medullary reticularformation (20). From our results and previous in vivostudies (19, 20), GABAergic inhibition appears to playa major role in th is system . Removal of th is inhibitionin vivo is suffic ient to activate circuitry that generatesmovement synergies ranging from walking and trottingto galloping. B locking GABAergic inhibition in vitrosim ilarly induced motor patterns for several locomotorgaits (F ig . 1, D an d E , confirm ing the presence of func-

t io na b c ir cu it ry f or g en er at io n of command signals forvarious movement synergies.

The locomotor circuitry in the spinal cord could bedirectly activated by certain excita tory am ino acids,dopam inergic agonists, or chobinergic agonists appliedto the spinal cord bath. The latency to onset of motoractivity after bath application of the agonists was 1-5mm . Dopam ine-induced activity was suppressed by theD2 receptor antagonists haloperidol (50-100 tM ). Anti-cholinesterase (25-50 M edrophonium) was requiredwith acetylcholine (20-50 tM ) to generate continuouslocomotor activity. Activ ity induced by acetybcholine oredrophonium was suppressed by the muscarin ic recep-tor antagonist atropine sulfate (10-50 ILM ). The loco-motor cycle period decreased with increasing neuro-chem ical concentration . The cycle period decreasedfrom 15.5 3.7to4.0 0.5s(n = 4)overtheeffectivedopam ine concentration range (0 .1-0.7 mM); itdecreased sim ilarly from 13.5 3.5 to 3.5 0.6 sn = 4) over the effective acetylchobine concentrationrange (10-50 tM in the presence of 50 LM edropho-


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Figure 2. Locomotor pattern generated by stim ulating som atosen-sory afferen t pathways by pinching base of tail in sp inal cord prepa-ration retaining lim bs and tail A an d B S ho rt d ura tio n (- 1 s) tailp inch induces transient bouts of h ind limb-forelim b locomotoractivity consisting of 5-30 locomotor cycles as illustrated in A byrecord ings of m otor d ischarge on th ird lum bar (L3) ventral roo ts.Average cycle period during locomotor ep isodes was typically1.5-2 .5 s. Sim ilar m otor patterns w ere elicited by electrical stim ula-tion (0 .2-m s pulses, 1-50 Hz, 10-200 teA , 5- to 30-s train duration)of single lumbar dorsal roots . B Patterns of ven tral root discharge(top traces) and hindlim b EMG (bo ttom traces) induced by sensoryactivation are sim ilar to walk ing patterns generated by chem icalstim ulation (Fig. I). R hythm ic m otor activ ity evoked by either sen-sory or chem ical stim ulation was suppressed by blocking sp inalNM DA receptors, suggesting involvem ent of sim ilar pattern gener-a tio n m e ch an is ms .

nium). Cycle frequency vs. agonist concentration rela-tionships for excitatory am ino acids are shown in F ig . 3.The motor output produced alternating steppingbikemovements of the hindlimbs at all cycle speeds w ithtypically an increase in the amplitude of the angulardisplacements of the limb joints as the level of excita-tion and cycle frequency increased. The cycle frequen-cies at the highest neurochem ical concentrations over-lap those w ith brain stem and sensory stimulation,implying sim ilar levels of spinal neuronal excitation .

Evidence was obtained that these neurochem icals in-duced motor activity by mim icking the action of rela tedendogenous neurochem icals on spinal neurons affectinglocomotor pattern generation. Nonselective neuronalexcitation was unlikely , because generalized depolariza-tion of spinal neurons produced by elevating extracellu larK concentration from 3 to 7 mM, enough to evokemotoneuronal discharge, d id not induce locomotor ac-tivity. To test for involvement of related endogenousneurochem icals, w e probed the system w ith neuro-chem ical uptake or breakdown inhibitors. The excita-tory am ino acid uptake inhibitor DHK (0.2-h mM)(F ig. 3) or antichobinesterase (50-200 tM edrophonium)applied to the spinal cord alone elicited motor patterns

for walking in a dose-dependent manner, indicating on-going endogenous release of excitatory am ino acids andacetylcholine whose synaptic concentrations can bebrought to threshold for activation of the locomotor cir-cuitry.

Earlier studies have shown that endogenous excitatoryam ino acids activating NMDA receptors6 are involvedin locomotor pattern generation in the spinal cord ofbower vertebrates (2 , 10-12). There is also evidence thatNMDA receptor activation mediates polysynaptic exci-tation in spinal sensorimotor pathways (18). W e there-fore characterized the receptor subtypes mediating theaction of the excitatory am ino acids. NMDA-receptoragonists (NMDA , L-aspartic acid , and L-glutam icacid) most effectively induced locomotor activity ; otherexcitatory am ino acids (kainic and quisqualic acids)were weakly effective or ineffective.6 The motor activityelicited by any of the am ino acids or their uptake inhib-itors was competitively inhibited by the selectiveN MD A-receptor antagonist D L-2-am ino-5-phosphono-valeric acid (AP5) (F ig . 3). The involvement of NMDAreceptors was further supported by the observationsthat: 1 an elevated spinal extracellular M g2 concen-tration (10 mM ) blocked locomotor activation by exoge-nous excitatory am ino acids,6 and 2 AP5 (10-100 tM )applied to the spinal cord bath suppressed the rhythm iclocomotor activity generated by activating brain-stemcircuitry or sensory stimulation (6 , 8), or by dopamin-ergic or cholinergic agonists. Therefore , all locomotoractivity ultimately depended on activation of spinalNM DA receptors.

DISCUSSIONThe results of this work establish the feasib ility of inves-tigating diverse aspects of the locomotor control systemin vitro including motor command and pattern genera-tion , as well as mechanism s for sensorimotor in tegration.In vitro brain stem and spinal cord are already routinelyused to investigate motor system s in lower vertebrates(2, 3, 22-24), and there have been efforts recently todevelop sim ilar in vitro mammalian preparations (5-8 ,9, 16, 25-29). The in vitro system developed in thesestudies is the first mammalian system shown to retainfunctional components of the brain stem -spinal cordlocomotor control circuitry. This system also retainsfunctional circuitry for other rhythm ic movements ofbrain stem -spinal cord origin (respiratory movements)

6Three classes of excitatory am ino acid receptors nam ed after theselective agonistsNMDA, kainic acid , an d quisqualic acid havebeen proposed tomediate theactionsofexcitatorymino acidneu-ro transm itters in the vertebrate CN S (18). NM DA receptor agonistsa re p ot en t activators of the spinal locomotor circuitry,hereasquisqualic acid (0.1 -100 toM ) applied to the sp inal cord (n = 4)produced only unpatterned motoneuron discharge. K ain ic acid(0 .75-5 sM ) induced rhy thm ic m otoneuron discharge in three ofsix preparations tested ; selective kainic acid receptor antagonistsare not availab le and pharmacological specificity could not beconfi rmed . NMDA receptors a re p o t en tl yb lo ck ed by M g2 e le va te dabove physiological concen trations of -1 mM (18).


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Figure 3. Involvement of excitatory am ino acids in locomotor pat-tern generation . NM DA , L -aspartic acid (L -ASP), and L-glu tam icacid (L-GLU) applied to spinal cord bath induce rhy thm icmotoneuronal discharge; d ischarge frequency increases w ithagonist concentration to a m axim um value beyond which the d is-charge becom es ton ic. NMDA alone induces continuous locomotoractivity lasting for several hours; th resho ld concen tration forNMDA is -3-4 iM w ith cycle frequency increasing over a narrowconcen tration range (- 3-7 MM ). D ischarge frequency vs. agonistconcentration relationships for L -ASP and L-GLU were obtainedwith am ino acid uptake inh ibito r DHK (0.2 mM ) in the bathingsolution; without DHK , L-GLU and L -ASP induce only transientep isodes of rhythm ic motoneuronal discharge. A fter a 15-mmpretreatm ent of the spinal cord w ith DHK , the motor discharge issteady for periods of up to several hours. DHK (0.2 mM ) aloneinduces low -frequency rhy thm ic discharge that is suppressed byNMDA receptor antagonist DL-2-am ino-5-phosphOflOValeric acid(AP5); higher discharge frequencies (not shown) can be induced byDH K in a dose-dependent m anner (0 .2-1 mM ). Thresho ld concen-trations for effects of L -GLU and L-ASP on cycle frequency in thepresence of DHK is -150 LM . Am ino acid dose-response relation-ships are displaced by AP5 , ind icating competitive antagonism andinvolvem ent of NMDA receptors in rhy thm generation .

(5, 7, 9), and it therefore appears that it has potentiallybroad application for investigation of major motor con-trol system s. Our demonstration of the viability ofmotor system circuits in vitro should encourage further

W e thank K . Kopec for assistance in the early experiments in th isseries, E . G arcia-R ill for suggesting the use of substance P to acti-vate brain-stem circu its, and R . Roy and S. Bodine for assistancewith EMG recordings. Supported by N ational Institutes of H ealth

NEURAL MECHAN ISMS FOR LOCOMOTION STUDIED IN VITRO 2287

investigation w ith this system into locom otor netw orksand other mechanisms involved in the neurogenesis ofmovement .

Our results suggest that diverse endogenous neuro-chem ical mechanism s underlie the generation of loco-motion, including peptidergic and GABAergic mecha-nism s in brain-stem networks, and dopam inergic ,cholinergic, and excitatory am ino acid-mediated mech-anism s in spinal cord networks. This diversity un-doubtedly reflects the multip le interacting networks ofthe motor command and pattern generation system s.The neuronal in terconnections and neurotransm ittersin these networks are unknown. The present resultsprovide evidence for several important endogenousneurochem ical mechanism s. The spinal networks receivemultip le inputs from bulbospinal and sensory afferentsystem s that may converge on a common set of spinalin terneurons critical for locomotor pattern generation(1). Our results are consistent w ith this hypothesis.Locomotor activ ity induced by sensory or brain-steminputs or by dopaminergic or cholinergic receptor acti-vation was suppressed by blocking NMDA receptors inth e spinal cord . These results suggest the follow ingpossibilities. 1 Bubbospinal, sensory afferent, anddopam ine-activated and acetylcholine-activated path-ways all release excitatory amino acids that activate acommon set of interneurons critical for locomotor pat-tern generation . If true, th is means that the descendingbulbospinal locom otor com mand signals a re tr an sm it te db y e xc it at or y am ino acids activating NMDA receptors.2 Activation of the spinal locomotor circuitry requirestonic (perm issive) inputs from interneurons releasing

.5 0 excitatory am ino acids t ha t a ct iv at e N MD A receptors.Our findings of a central ro le for endogenous excita-

tory amino acids activating NMDA receptors in neuro-genesis of locomotion extend sim ilar findings for bowervertebrates. Rhythmic locomotor movements rangingfrom undulatory swimming in primitive cyclostomes(2, 3) to walking by tetrapod mammals now appear todepend on this excitatory am ino acid-mediated mecha-nism , which suggests a phylogenetic conservation ofthis m echanism for rhythm ic motor pattern generation(5 , 13). These locomotor pattern generation systemsconsist, in p ar t, o f o sc il la ti on -g en er at in g c ir cu it ryproducing the locomotor cycle rhythm (1-4). The dataindicate that excitatory am ino acids are importan t foractivating this component (Fig. 3). Inasmuch asNMDA-receptor activation can induce bistable n euron almembrane properties, these receptors may also bedirectly involved in the generation of oscillatory activity(15). However, the neurons activated by the excitatoryam ino acids and the relation between their activity andrhythmogenesis have not been determ ined in any ver-tebrate system . The in vitro system developed hereshould provide the opportunity to determ ine the under-ly ing neuronal mechanism s in the mammalian system .


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g ra nt s N S- 24 74 2, H L- 23 82 0, an d H L-37941. J. C . Sm ith is aParker B . Francis Foundation Fellow . B . J. Schm idt is supported bya fellow ship from the Medical Research Council o f Canada.

REFERENCES1. GRILLNER, S. C ontrol of locom otion i n b ip ed s, t et ra -pods, and fish . Brookhart,J. M .; Mountcastle, V . B .;Kandeb, E . R ., eds. Handbook of physiology,Section1,

Th e nervous system , Vol. II, Motor control,Part 2.Bethesda: American Physio logical Society ; 1981:1179-1236.2. GRILLNER, S .; WALLN, P. Central pattern generatorsfor locomotion, w ith special reference to vertebrates.A nn u. R ev . N eu ro sc i.8: 233-261; 1985.3. GRILLNER, S. Neurobiological bases of rhythm ic motoracts. Science 228: 143-149; 1985.4. FELDMAN, J. L.; GRILLNER, S . Control of vertebraterespiration and locomotion: a brief account. Physiologist26: 310-316; 1983.5. SMITH, J. C.; FELDMAN, J. L. M otor patterns for respi-ra tion and locomotion generated by an in vitro brain-stem -spinal cord from neonatal rat. S oc. N euro sci.Abstr. 11: 24; 1985.6. SMITH,J . C.; GARCIA-RILL, E.; FELDMAN,J . L . C hem i-cal activation of mammalian locomotion in vitro. Soc.Neurosci . Abstr. 12: 386; 1986.7. SMITH,J. C.; FELDMAN,J . L . In vitro b rainste m-spinalcord preparations for study of motor systems for mam -m alian respiration and locom otion.]. N eu ro sc i. M e th od s21: 321-333; 1987.8. SMITH,J . C.; FELDMAN,J . L. Neural control system formammalian locom otion in v itro. Soc. N eurosci. A bstr.13: 825; 1987.9. SUZUE, T. Respiratory rhythm generation in the in vitrobrain stem -spinal cord preparation of the neonatal rat.J. Physiol. London 354: 173-183; 1984.

10 . BRODIN , L.; GRILLNER, S. The role of putative excitatoryam ino acid neurotransmitters in the initiation of loco-motion in the lamprey spinal cord. I. The effects of ex-citatory am ino acid antagonists. Brain Res. 360:139-148; 1985.11 . BRODIN , L.; GRILLNER, S. The role of putative excitatoryam ino acid neurotransmitters in the initiation of loco-motion in the lamprey spinal cord. II. The effects ofam ino acid uptake inhibitors. Brain Res. 3 60 : 1 49 -1 58 ;1985.12 . DALE, N .; ROBERTS, A . E xcitatory am ino-acid receptorsin Xenopus embryo spinal cord and their robe in theactivation of sw imm ing. j Physiol. London 348:527-543; 1984.13 . DALE, N . Excita tory synaptic drive for sw imm ing medi-ated by am ino acid receptors in the lamprey. J. Neuro-sd . 6: 2662-2675; 1986.

14 . JoRD AN , L. M . Factors determ ining m otoneuron rhyth-micity during fictivelocomotion. S ym p. S oc . E xp . B io l.37: 423-444; 1983.

15 . WALLN, P .; GRILLNER, S . N -Meth yl -D -a sp ar ta tereceptor-induced, inherent oscillatory activ ity in neu-rons active during fictive locom otion in the lamprey.].Neurosci . 7: 2745-2755; 1987.16 . KUDO, N.; YAMADA, T . N -M ethyb-D ,L -asparta te inducedlocomotor activity in a spinal cord-hindlimb musclespreparation of the newborn rat studied in vitro. Neuro-s ci. L ett. 75: 43-48; 1987.17 . PERRET, C . Centrally generated patterns of m otoneuronactiv ity during locomotion in the cat. S ym p. S oc . E x p.Biol. 37: 405-422; 1983.

18 . MAYER, M .; WESTBROOK, G. The physiology of excita-tory am ino acids in the vertebrate central nervous sys-tem. Prog. Neurobiol.28: 197-276; 1987.

19 . GARCIA-RILL, E.; SKINNER, R. D .; F ITZGERALD, J. A .Chem ical activation of the mesencephabic locomotorregion. Brain Res. 330: 43-54; 1985.20 . GARCIA-RILL, E. The basal ganglia and the locomotorregions. Brain Res. Rev. 11: 47-63; 1986.21 . CONRAD, C.; HENDERSON, V .; SKINNER, R. D .; ABRA-

HAM, P .; GARCIA-RILL, E. Development of the pedun-culopontine nucleus. I. In vivo. Soc.Neu rosci.Abstr. 13 :1 17 6; 1 98 7.

22 . GRILLNER, S .; WALLN , P. ; MCCLELLAN, A.; SIGvARDT,K .; WILLIAMS, T.; FELDMAN,J . L. The neural genera-tion of locomotion in the lam prey: an incomplete ac-count. S ym p. S oc . E xp . B io l. 37: 285-303; 1983.23 . ROVAINEN, C . M . Generation of respiratory activ ity bythe lamprey brain exposed to picrotoxin and strychnine,and weak synaptic inhibition in motoneurons. Neuro-science 10: 875-882; 1983.24 . MCCLELLAN, A . D . Descending control and sensorygating of fictive swimm ing and turning responseselicited in an in vitro brainstem /spinal cord prepara-tion ,of the lam prey. B rain R es. 302: 151-162; 1984.25 . LLINAS, R.; YAROM , Y.; SUGIMORI , M. Isolated mam -malian brain in vitro : new technique for analysis ofelectrical activ ity of neuronal circu it function . Federa-tionProc. 40: 2240-2245; 1981.

26 . BAGUST,J.; KELLEY, M . E. M .; KERKUT, G . A. An iso-lated mammalian brainstem -spinal cord preparationsuitable for the investigation of descending control ofm oto r a ctiv ity . Brain Res. 327: 370-374; 1985.27 . RICHERSON, G . B.; GErF INO, P. A . Respiratory activityin a perfused guinea pig brain/spinal cord preparation .Soc. Neurosci.Absir. 10: 745; 1984.28 . SHAPOvALOv , A. I.; SHIRIAEv, B . I.; TAMAROVA, Z. A .A study of neuronal activity of mammalian superfusedor intra-arterialby perfused C NS preparations. Kerkut,G . A .; Wheal, H . V ., eds. E le ctr op hy sio lo gy o f is ola tedmammal ian CN S preparat ions. New York: Academ ic;1 98 1: 3 76 -3 94 .29 . WALTON, K.; LLINS, R. An isolated in vitro prepara-tion of the neonatal rat brainstem and spinal cord . Soc.N eu ro sci. A bs tr . 11: 24; 1985.

R ec eiv ed fo r p ub lic atio n O cto be r 1 6 1987.Accepted for publication January 13, 1988.

Neural Mechanisms Generating Locomotion in Mammals

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