Effects of Intrathecal Glutamatergic Drugs on Locomotion I. NMDA in

J Neurophysiol 88: 3032–3045, 2002;
Effects of Intrathecal Glutamatergic Drugs on Locomotion
I. NMDA in Short-Term Spinal Cats
Centre de Recherche en Sciences Neurologiques, Faculté de Médecine, Université de Montréal, Montreal, Quebec H3T 1J4;
School of Physical and Occupational Therapy, McGill University, Montreal, Quebec, H3G 1A5; and 3Department of
Physiology, University of Manitoba, Winnipeg, Manitoba, R3E 0W3, Canada
Received 25 February 2002; accepted in final form 2 August 2002
Address for reprint requests: S. Rossignol, Centre de Recherche en Sciences
Neurologiques, Faculté de Médecine, Université de Montréal, Pavilion PaulG.-Desmarais, 2900 Chemin de la Tour, Montréal, Québec H3T 1J4, Canada
(E-mail: [email protected]).
long-term chronic spinal cats will be addressed in a forthcoming
companion paper.
The spinal cord contains a neural circuitry capable of generating rhythmic locomotor pattern in the absence of any
peripheral or descending inputs (Grillner 1981). Such neural
circuitry, commonly known as central pattern generators
(CPG), has been studied in detail in lower vertebrates such as
the lamprey (Brodin and Grillner 1986a,b). It comprises a few
components including excitatory interneurons and mutually
inhibitory interneurons. While N-methyl-D-aspartate (NMDA)
is primarily responsible for mediating the excitatory component of the CPG (Brodin et al. 1985; Buchanan and Grillner
1987), glycine is found to be primarily responsible for mediating the reciprocal inhibition.
In normal cats, the CPG has been shown to be “activated” by
descending inputs (such as the reticulospinal tract) and acts as
a command center to generate goal-oriented and anticipatory
adaptive changes to locomotion (for review, see Rossignol
1996). In lamprey, it has been shown that the reticulospinal
tract that “drives” the CPG activates NMDA and non-NMDA
receptors (Buchanan et al. 1987). Peripheral afferents that also
utilize excitatory amino acids (EAA) as neurotransmitter also
interact with the CPG to adjust the locomotor pattern for
corrective changes. Thus the CPG is in constant interaction
with both the sensory and supraspinal input to adapt the locomotor pattern to changes in the environment.
Exogenous applications of pharmacological agents that
mimic the neurotransmitter of the descending pathways and act
on receptors of intraspinal neurons have also been shown in
different preparations to be effective in either triggering and/or
modulating locomotion. EAA has been reported to induce
locomotion in different in vitro preparations mainly the lamprey (Brodin and Grillner 1985a,b; Cohen and Wallen 1980;
Grillner et al. 1981; Poon 1980). The receptor subtypes for
mediating the action of the EAA were found to be NMDA and
non-NMDA receptor, kainate but not quisqualate. It appears,
however, that NMDA and kainate plays a different role in
locomotion. For example, in reduced preparations in which
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0022-3077/02 $5.00 Copyright © 2002 The American Physiological Society
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Chau, Connie, Nathalie Giroux, Hugues Barbeau, Larry Jordan,
and Serge Rossignol. Effects of intrathecal glutamatergic drugs
on locomotion. I. NMDA in short-term spinal cats. J Neurophysiol 88:
3032–3045, 2002; 10.1152/jn.00138.2002. Excitatory amino acids
(EAA) have been reported to induce fictive locomotion in different in
vitro and in vivo preparations in a variety of species through their
actions on both N-methyl-D-aspartate (NMDA), and non-NMDA receptors. NMDA-induced intrinsic membrane properties such as intrinsic motoneuronal membrane oscillations and plateau potentials have
been suggested to play a role in the generation of locomotion. There
is, however, no information on the ability of NMDA in triggering
spinal locomotion in awake behaving animals. Because most of the
previous work on the induction of locomotion has concentrated on
monoaminergic drugs, mainly noradrenergic drugs, the aim of this
study is to examine the potential of NMDA in initiating locomotion in
chronic spinal cats within the first week after spinalization. Five cats
chronically implanted with an intrathecal cannula and electromyographic (EMG) electrodes were used. EMG activity synchronized to
video images of the hindlimbs were recorded. The results show that
during the early posttransection period (within the 1st week postspinalization), NMDA did not trigger robust locomotion as did noradrenergic drugs. The predominant effects of NMDA were a general
hyperexcitability reflected by fast tremor, toe fanning, and an increase
in small alternating hindlimb movements with no foot placement nor
weight support. During the intermediate phase posttransection (6 – 8
days), when the cats were able to make some rudimentary steps with
foot placement, NMDA significantly enhanced the locomotor performance, which lasted for 24 –72 h postinjection. NMDA was also
found to increase the excitability of the cutaneous reflex transmission
only in early spinal cats. One possible hypothesis for the ineffectiveness of NMDA in triggering locomotion in early spinal cats could be
attributed to the widespread activation of NMDA receptors on various
neuronal elements involved in the transmission of afferent pathways
that in turn may interfere with the expression of locomotion. The
marked effects of NMDA in intermediate-spinal cats suggest that
NMDA receptors play an important role in locomotion perhaps
through its role on intrinsic membrane properties of neurons in shaping and amplifying spinal neuronal transmission or by augmenting the
sensory afferent inputs. The long-term effects mediated by NMDA
receptors have been reported in the literature and may involve mechanisms such as induction of long-term potentiation or interactions
with neuropeptides. The effects of NMDA injection in intact cats and
J Neurophysiol • VOL
and Rossignol 1991; Chau et al. 1998a,b). Noradrenergic drugs
(L-DOPA and clonidine), but not serotonergic or dopaminergic
drugs, were found to trigger treadmill locomotion in chronic
spinal cats during the first week after spinalization (Barbeau
and Rossignol 1991). Typically, at 2–3 days postspinalization,
before any drug injection, the spinal cat has very little hindlimb
movement when placed on a moving treadmill belt. In these
preparation, injection of noradrenergic drugs triggered wellcoordinated locomotion with weight support and plantar paw
placement in cat (Barbeau and Rossignol 1991; Barbeau et al.
1987; Chau et al. 1998a,b). This marked locomotor effect was
seen within minutes when clonidine (an ␣2 agonist) was given
intrathecally in a 3d-spinal cat (Chau et al. 1998b).
To date, NMDA receptors appears to be the primary EAA
receptors necessary for locomotion. There is, however, no
report on the effect of NMDA in triggering locomotion in
awake spinal cats. The present study was undertaken to examine the ability of EAA to trigger locomotion in adult chronic
spinal cat shortly after spinalization (within 10 –12 days). Another paper will deal with the modulation of locomotion in the
intact cat as well as the chronic spinal cat by the same agonist
and antagonists of NMDA receptors (N. Giroux, C. Chau, H.
Barbeau, T. A. Reader, and S. Rossignol, unpublished data).
NMDA was found to have limited effects on locomotion in
both intact and long-term spinal cats. AP5, an NMDA receptor
antagonist, somewhat disturbed locomotion in intact cats by
reducing weight support but altogether abolished locomotion in
spinal cats, suggesting the importance of glutamatergic system
in mediating locomotion in chronic cats. Some parts of this
work have been published in abstract form (Chau et al. 1994).
Five adult cats, previously trained to walk on a treadmill were used
for this study. We examined the effects of NMDA in these cats during
the early postspinalization period (shortly after spinalization where
there was no locomotion, usually within 6 days postspinalization) and
intermediate postspinalization period (period where there were few
rudimentary steps with inconsistent foot placement, usually around
7– 8 days postspinalization). Thus in the intermediate period, there
were only limited locomotor movements such as the attempts of foot
placement, and an increase in the ability to generate consecutive
rhythmic steps even though the steps were very small compared with
normal. During the experiments, treadmill locomotion and responses
to cutaneous stimulation were recorded before drug injection and at
different times intervals after each intrathecal drug injection.
The effect of NMDA was tested in four cats (CC6, CC7, NG3, and
NG5) during the early-postspinalization period and was tested in three
cats (NG2, NG3, and NG5) during the intermediate-postspinalization
period. All cats were chronically implanted with EMG electrodes and
an intrathecal cannula. In four cats (CC6, CC7, NG2, and NG5), nerve
cuff electrodes were also chronically implanted on the superficial
peroneal nerve for reflex testing. The pre- and postdrug trials were
carried out on the same day and could be compared with normal
treadmill locomotion of the same cat (a within subject design where
each animal has its own baseline for comparison).
A detailed description of the surgeries has been made elsewhere
(Chau et al. 1998a,b). Briefly, all surgeries were carried out in aseptic
conditions, and all procedures followed a protocol approved by the
Ethics Committee of Université de Montréal.
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locomotor activity was recorded from motor nerves, activation
of NMDA receptor induced a locomotor pattern (fictive locomotion) which resembles the electromyographic (EMG) pattern during actual swimming in intact lamprey (Brodin and
Grillner 1985a) and tadpole (Dale and Roberts 1984). Activation of kainate receptors elicited a much faster swimming in
both lamprey and tadpole, and the induced swimming activity
was also less regular than NMDA-induced swimming (for
review, Grillner 1986).
In an isolated brain stem-spinal cord preparation of neonatal
rats, dopamine alone, EAA (L-aspartic or L-glutamic acid), or
NMDA was effective in evoking locomotor activity recorded
as rhythmic ventral root discharges (Smith and Feldman 1987).
NMDA receptors were most effective in inducing locomotor
activity; other non-NMDA receptors (kainate and quisqualate)
were weakly effective or ineffective (Smith et al. 1988). In
another spinal cord-hindlimb neonatal rat preparation, bath
application of N-methyl-D,L-aspartate (NMA) elicited alternating rhythmic activity in extensor and flexor muscles that was
completely blocked by NMDA receptor antagonist, 2-amino5-phosphonovaleric acid (AP5) (Kudo and Yamada 1987).
NMDA alone or together with serotonin was also effective in
producing a robust locomotor rhythm in neonatal rats (Cazalets
et al. 1992; Cowley and Schmidt 1994; Kiehn et al. 1996).
Recent study shows that even in the absence of action potential, by removing extracellular Ca2⫹ or adding tetrodoxin
(TTX) to the perfusion medium, a combined NMDA and 5HT
was still effective in triggering stable locomotor activity measured by ventral root activity in neonatal spinal cord (Tresch
and Kiehn 2000).
In Xenopus embryos, bath application of NMDA or kainate
caused a sustained motor output pattern similar to swimming
evoked by natural stimulation of the intact animals (Dale and
Roberts 1984). Recent studies show that excitatory premotor
interneurons play a major role in determining the frequency of
the swimimng CPG through glutamatergic synaptic drive (Sillar and Roberts 1993; Zhao and Roberts 1998). In in vitro
spinal cord of adult spinal frogs, only NMA was effective in
eliciting locomotor activity (McClellan and Farel 1985). In
decerebrate and spinal rabbits, systemic injection of MK-801,
an NMDA-channel blocker, dose-dependently blocked the
spontaneous and L-DOPA-induced fictive locomotion (Fenaux
et al. 1991). In a brain stem-spinal cord preparation from an
adult urodele, fictive rhythmic motor patterns were evoked by
bath application of NMDA with D-serine (Delvolve et al.
In acute in vivo preparation such as the decerebrate cats,
NMDA was also found to produce a well-coordinated fictive
locomotor pattern (Douglas et al. 1993). In these decerebrate
cats, intrathecal administration of NMDA was found to elicit
hindlimb fictive locomotion similar to that evoked by the
mesencephalic locomotor region (MLR). Kainate and quisqualate were ineffective in producing fictive locomotion in
these decerebrate cats (Douglas et al. 1993). In acutely spinalized marmoset monkey, clonidine evoked alternating fictive
locomotor activity in all monkeys, whereas NMDA or NMA
were found to evoke rhythmic alternating nerve activity in 7 of
10 monkeys (Fedirchuk et al. 1998).
In awake chronic preparations, the ability of EAA to initiate
locomotion is not known. Previous work on spinal cats have
mainly dealt with the role of monoaminergic drugs (Barbeau
In four cats (CC6,
CC7, NG2, and NG5), custom-made bipolar cuff electrodes made
from polymer (Julien and Rossignol 1982) (⬃1 cm length) were used
to stimulate the superficial peroneal nerve (⬃6 mm between electrodes leads) through a 2-pin-head connector.
A laminectomy was performed at the Th13 vertebra. At the level of transection, local anesthetics [lidocaine hydrochloride (Xylocaine), 2%] was placed on the spinal cord (a few drops)
and injected into the spinal cord directly (⬃200 ␮l injections on each
side). The intrathecal cannula was identified, and care was taken to
avoid any damage during the complete severance of the spinal cord
with a micro scissor. Once the bottom of the vertebral spinal canal
could be clearly visualized, an absorbable hemostat (Surgicel, oxidized regenerated cellulose) was used to fill the space between the
rostral and caudal ends of the spinal cord. The completeness of the
spinal transection was later confirmed with histological analysis
(10-␮m serial sections using the Kluver-Barrera method).
Animal cares
treadmill), and only locomotion of the hindlimbs was recorded. A
Plexiglas separator was put between the hindlimbs to prevent crossing
of the hindlimbs due to increased adductor tonus often seen in spinal
cats (Barbeau and Rossignol 1987). At this early postspinalization
period, the experimenter also needed to lift the tail of the cat to
support the weight of the hindquarters of the cat and to provide
Video images of the locomotor movements were captured by a
digital camera (Panasonic 5100, shutter speed 1/1,000 s) and recorded
on a video recorder (Panasonic AG 7300). Reflective markers were
placed on the bony landmarks of the left hindlimb facing the camera:
the iliac crest, the femoral head, the knee joint, the lateral malleolus,
the metatarsal phalangeal (MTP) joint and the tip of the third toe
during each session. Additional markers were also placed on the trunk
of the cat for calibration (10 cm). Recorded video images were
digitized semi-automatically with a temporal resolution of 16.7 ms
using the 2D PEAK Performance system (Peak Performance Technologies, Englewood, CA). Displacement data and angular joint
movements were calculated from the digitized x-y coordinates and
could be displayed as continuous angular displacements (running
averages of 5 values) for a normalized step cycle or as stick diagrams.
EMG data recorded during locomotion were displayed on an electrostatic polygraph (Gould, Model ES 1000), and a segment representative of the animal’s performance was used for analysis. All
EMGs were differentially amplified with a bandwidth of 100 Hz to 3
kHz and recorded on a 12-channel video recorder (Vetter Digital,
model 4000A PCM recording adapter) with a frequency response of
1.2 kHz per channel. The EMG signals were digitized at 1 kHz,
rectified, (using St as the onset of the cycle), and averaged over a
number of cycles.
Also, a digital SMPTE (Society for Motion Picture and Television
Engineers) time code generated by a Skotel time code generator
(model TCG-80N) was simultaneously recorded on the EMG tape and
was inserted into the video images thus allowing synchronization of
video images and EMG data.
Single pulse of 250 ␮s duration was
delivered (Grass S88 stimulator) at 0.4 – 0.5 Hz through the cuff
electrodes. The stimulation was given either at rest, standing, or
sitting. The stimulus signal was displayed on an oscilloscope together
with selected EMGs. The threshold (T) of the stimulation was determined by observing a just detectable response in St at rest. The EMG
responses to the electrical stimulation was also digitized at 1 kHz, and
computer averaged.
Drug applications
Animals were placed in an incubator until they regained consciousness after surgery. They were then housed in spacious individual
cages (104 ⫻ 76 ⫻ 94 cm) with food and water. In the first postop
day, torbugesic (Butorphanol tartrate, 0.05 mg/kg sc, every 6 h) was
given. After spinalization, cats were placed in cages lined with foam
mattresses and were attended to a few times daily to check and clean
the head connectors, to flush the intrathecal cannula with sterile saline,
to express the bladder manually, and to generally inspect and clean the
Recording procedures and protocol
LOCOMOTION. All cats were placed on the treadmill and locomotion
was recorded a few days after the intrathecal catheterization and the
implantation of EMG electrodes and/or nerve cuff electrodes. Locomotion at different speeds was recorded while the cats walked freely
on the treadmill belt. This served as the baseline controls (the intact
trials) for each cat and was recorded for 6 –7 days for CC6 and CC7
and for 7, 11, and 4 days for NG2, NG3, and NG5, respectively.
To evaluate treadmill locomotion after spinalization, the forelimbs
of the spinal cat were placed on a platform (⬃2 cm above the
J Neurophysiol • VOL
Three drugs were used in these experiments. They are NMDA, an
EAA receptor agonist; dihydrokanic acid (DHK), an EAA uptake
blocker, and clonidine (2,6,-dichloro-N-2 imidazolidinylid-enebenzenamine), ␣2-noradrenergic agonist (all drugs are from Sigma). All
drugs were dissolved in sterile saline solution and were injected as a
bolus into the spinal cord through the intrathecal cannula. All bolus
injections were of 100 ␮l per dose followed by a subsequent bolus
injection of saline (approximately 100 –150 ␮l) to fill the dead space
of the cannula and ensure the expulsion of the drug into the intrathecal
space of the spinal cord. NMDA (0.1–10 mM it) were given to CC6
and CC7 in multiple injections in attempt to trigger locomotion in the
early postspinalization phase. NMDA (1 mM it) was given to NG2,
NG3, and NG5 at different days postspinalization. NMDA was injected at 3 days for CC6 and at 3, 4, 5, and 6 days for CC7. DHK (5
mM it) was also given to CC7. NMDA was injected at 7 and 11 days
for NG2; at 6 and 8 days for NG3; and at 5, 6, 7, and 8 days for NG5.
The upper limit of liquid volume given in one session was ⬃800 ␮l
over 4 –5 h. It should be noted that the dose range that we injected in
the spinal cat was much smaller than that reported to cause spinal cord
damage in chick embryo (Llado et al. 1999). Also, the chronic spinal
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CATHETERIZATION. The intrathecal cannulation
technique (Chau et al. 1998b). was adapted from the procedure of
Espey and Downie (1995), A midline incision from the cranium to
C2–3 level was made and a Teflon tubing (24LW) was inserted
through the cisterna magna down to approximately L4–L5 spinous
processes. The other end of the Teflon tube, previously connected to
a cannula connector pedestal (Plastic One) was secured on the skull
with acrylic cement as a port of entry. The location of the cannula tip
on postmortem examination are as follows: L5 (right side, ventrolateral) for CC6; L4 –5 (right side, ventrolateral) for CC7; L3– 4 (central,
dorsolateral)for NG2; L2 (dorsolateral) for NG3; and L4 (ventrolateral
to dorsal rootlets) for NG5.
IMPLANTATION OF EMG ELECTRODES. Four cats were implanted
with two 15-pin-head connectors (TRW Electronic Components
Group) while one cat was implanted with only one connector secured
to the cranium using acrylic cement. A pair of stainless steel wires
were sewn into the belly of selected muscles with the Teflon coating
removed at the point of insertion. One unpaired wire was placed
deeply under the skin of the neck and served as an electrical ground.
Bilaterally implanted muscles include iliopsoas (Ip), a hip flexor;
sartorius anterior (Srt), a hip flexor and knee extensor; semitendinosus
(St), a knee flexor and hip extensor; vastus lateralis (VL), a knee
extensor; gastrocnemius lateralis (GL), an ankle extensor and knee
flexor; tibialis anterior (TA), an ankle flexor; and gastrocnemius
medialis (GM), mainly an ankle extensor.
cats were kept for a long time, and we could not observe any obvious
deterioration of the locomotor performance of the cat that would have
resulted from a mechanical or toxic damage to the cord.
ization) to test the effect of NMDA on a preexisting but very
rudimentary locomotor pattern. Video records of each trial
were carefully reviewed, and representative trials were selected
for detailed quantitative kinematic and EMG analysis.
The results are from experiments performed in five chronic
spinal cats. To test the ability of NMDA in initiating locomotion soon after spinalization, different doses of NMDA (100 ␮l
of 0.1–10 mM) were injected in four cats soon after spinalization (starting at 3 days postspinalization in cats CC6 and CC7,
at 6 days in cat NG3, and at 5 days in cat NG5). In three cats,
NG2, NG3, and NG5, a fixed dose of NMDA (1 mM it) was
also injected at an intermediate stage (⬃6 –7 days postspinal-
Effects of intrathecal NMDA on locomotion in the early
postspinalization phase
NMDA alone or potentiated with DHK did not trigger locomotion in four spinal cats (total of 7 injections) during the
early postspinal days (3– 6 days). Figure 1A shows a typical
example of a 3d-spinal cat (CC7) before NMDA injection.
Thirty minutes after the NMDA injection (1 mM it, Fig. 1B),
some limited rhythmic movements of the hindlimbs were seen
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FIG. 1. The effects of N-methyl-D-aspartate (NMDA) on the initiation of locomotion in an early spinal cat (3 days, CC7).
A: stick diagram (1 step cycle) and raw electromyographic (EMG) traces of hindlimb flexors (Srt, St) and extensor (VL) of the left
(L) and right (R) sides during treadmill locomotion before any drug injection. B: locomotion recorded at 30 min after NMDA
(1 mM it). C: locomotion recorded at 16 min after clonidine (4 mM it) injection. Clonidine was injected 2 h after the NMDA
injection. An organized locomotor motor pattern, with plantar foot placement and weight support of the hindquarters, was observed.
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Effects of intrathecal NMDA on locomotion in the
intermediate phase postspinalization
While NMDA has been shown repeatedly not to be able, a
few days after spinalization, to trigger robust locomotion in
spinal cats contrary to a noradrenergic agonist such as clonidine, NMDA significantly ameliorated the locomotor pattern
in all three spinal cats (5 injections) that had already regained
the ability to generate rudimentary stepping with occasional
foot placement but little weight support, usually around 6 – 8
days postspinalization. Figure 4 shows that before NMDA
injection, a 7d-spinal cat (NG2) was only capable of making
very small but regular steps (stance length ⫽ 43 ⫾ 18 mm,
13.7% of intact values) with a toe contact (not plantar) at the
onset of stance as noted by the horizontal bar in the stick
diagram (Fig. 4A). The EMG traces show disorganized but
occasional burst activity of knee flexors (St) and very weak hip
flexor activity (Srt). At 17 min after NMDA injection (1 mM
it), significant changes in the locomotor pattern was observed
as shown in Fig. 4B. There was a marked increase in EMG
activity such as the hip flexor (Srt), knee flexor (St), and ankle
extensors (GL and GM). The foot placement also improved as
seen in the stick diagram at the beginning of the stance phase
(indicated by a shaded bar). The locomotor pattern was robust
and regular with marked increase in step length (almost 4 times
the pre-NMDA value), step cycle duration, joint angular excursion, and weight support. In addition, the cat was able to
walk ⱕ0.6 – 0.7 m/s (see Fig. 5B), whereas it could only cope
at 0.2– 0.3 m/s before NMDA injection. The effect of NMDA
persisted for 24 and 72 h after NMDA injection as shown in
Fig. 4, C and D, respectively. Despite some deterioration in the
locomotor pattern over time, namely a decrease in the activity
of flexor muscle (St, Srt) and especially the extensor muscle
(GL, GM), the spinal cat was still able to generate some
stepping on the treadmill.
A further examination of the EMG activities after NMDA
injection in the intermediate phase revealed a marked increase
in the flexor (St) burst duration as shown in Fig. 5. Because
there was little locomotion in the spinal cat before giving the
drug, quantitative analysis of the EMG pattern was limited to
locomotion after NMDA injection where a clear EMG bursting
pattern can be observed. These were then compared with the
readings of trials in the same cat in the intact state. Typically,
the burst duration of the flexor muscle (St) during locomotion
was much shorter than that of the extensor muscle (GL) in
normal cats. After spinalization, the locomotor pattern observe
after NMDA injection is characterized by a marked increase in
the flexor burst duration while the extensor burst increase to a
lesser extent, thus the ratio between flexor and extensor muscle
activation was changed. The numeric values of the muscle
burst duration of the intermediate-spinal cat studied was shown
in Table 1. Note that the increase in the flexor (St) burst
duration is more marked than that of the extensor (VL, GL).
In addition, at 24 h after NMDA injection, the ankle extensor
(GL) burst duration and the step cycle duration were found to
decrease as the speed of the treadmill increases, indicating the
improved ability of the cat (NG2) to adapt the locomotor
pattern to increasing treadmill speeds (0.6 – 0.7 m/s). This is in
contrast to the observation in the same cat before NMDA
injection where it can only cope with a maximum treadmill
speed of 0.2– 0.3 m/s.
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but with no EMG activity from knee extensors. Because there
were very little movements from the hip, the hindlimb remained in an extended position. There was therefore neither
plantar foot placement nor weight acceptance. After an additional dose of NMDA (5 mM it; results not shown), the already
faint rhythmic movements of the hindlimbs actually decreased.
This limited effect of NMDA was sharply contrasted by the
effects of a noradrenergic agonist, clonidine. Indeed a subsequent clonidine injection (Fig. 1C) elicited a robust and wellcoordinated locomotor pattern with alternating flexor and extensor burst activities. The cat was also capable of plantar foot
placement, good weight support during stance as can be seen
from a marked increase in knee extensor EMG activities (Fig.
1C), and could walk ⱕ0.5 m/s. Therefore the inability of the
previous NMDA injection in triggering locomotion could not
be attributed to the inability of the cat to generate spinal
A higher concentration of NMDA (2 consecutive doses of
NMDA, 5 mM it each given 30 min apart) given in another 3
days spinal cat (CC6) still did not initiate any organized locomotion but did produce sporadic spontaneous high-frequency
rhythmic movements (10 Hz) which lasted for bouts of of 0.5
to 1 s as shown in Fig. 2. It consisted of a high-frequency burst
of flexor and extensor activity. The spontaneous high-frequency movements were also recorded in another spinal cat
(CC7) where a higher concentration of NMDA was given.
In one cat, (CC7) NMDA was capable of initiating rhythmic
stepping movements but with very little weight support at 4
days postspinalization as shown in Fig. 3. While there was no
foot placement before NMDA (Fig. 3A), 7 min after the first
NMDA injection (1 mM), there was a marked increase in
stepping with plantar foot placement on the left hindlimb (Fig.
3C). On the right hindlimb (results not shown), only the tip of
the toes touches the treadmill belt with no dorsiflexion at the
ankle (toe placement). These stepping movements were very
different from the locomotor pattern triggered after clonidine
injection on the previous day. First, the stepping was only
triggered with maximal perineal stimulation and was asymmetrical. While the left limb showed consistent plantar foot placement and alternate EMG burst activities (Fig. 3D), the right
limb had inconsistent toe placement, and much weaker EMG
activities (Fig. 3B). Second, the locomotor pattern also fatigued
easily. Typically, the pattern became disorganized after only a
few cycles. Third, there was also very little weight support
from the cat and the experimenter had to support most of the
weight of the cat, as could be seen in the low activities on
extensor muscles, especially the left ankle extensor (LGL).
Last, the cat could only cope with treadmill speed ⱕ0.2m/s.
With an additional dose of NMDA (1 mM) the maximal
treadmill speed that the cat could cope with was 0.3 m/s.
In summary, NMDA injection did not initiate robust locomotion as did the noradrenergic agonist, clonidine. In most
cases, the maximal effect after NMDA injection was an increase in some alternating movement of the hindlimbs but
without any plantar foot placement nor any weight acceptance
during stepping. In one case (CC7), NMDA elicited stepping
with foot placements, but these steps were asymmetrical, transient, limited to low treadmill speed, and with very little weight
acceptance during stance.
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FIG. 2. The effects of high concentration of NMDA in a 3 days spinal cat (CC6). A: raw EMG traces of hindlimb muscles during treadmill locomotion (0.2
m/s) before drug injection. No locomotion can be elicited even with strong perineal stimulation. B: recordings taken at 9 min after a 2nd dose of NMDA (5 mM
it) given at 30 min after the 1st dose (5 mM it), multiple episodes of spontaneous paw-shake activity (1) was observed when the cat was at rest (lying on a foam
mattress). C: expanded portions of one paw-shake episode indicated by 1 in B.
Long-term effects of NMDA
The effects of NMDA lasted for a long time ranging from
2– 4 days after injection. A detailed evolution of the locomotor
performance in 1 spinal cat (NG2) is shown in Fig. 6. The cycle
duration was measured as an index of the effects of the drugs
as well as an indirect indication of the quality of the locomotion. As shown in the figure, the spinal cat had received NMDA
at 7 days and later at 11 days postspinalization (indicated by
the arrows). After NMDA injection at 7 days, a marked increase in cycle duration was seen at 17 min after injection (⽧)
as compared with cycle duration before NMDA injection (〫).
The cycle duration after NMDA injection also approached the
values previously obtained in the intact state as represented by
the shaded band. Note that even after 24 h after NMDA
injection, there was still a marked increase in the cycle duration
(771 ⫾ 64 ms) as compared with before NMDA injection
(533 ⫾ 80 ms). By 72 h (10 days), the locomotor pattern began
to deteriorate and was characterized by a decrease in weight
support, less consistent plantar foot placement during stance,
and decreased step cycle duration. A second NMDA injection
was given to the cat on the following day (11 days). Again, a
marked increase in cycle duration was still observed 24 h after
the injection (12 days). The effect of NMDA diminished with
time as previously observed and by 14 days, the cycle duration
was comparable to 11 days, before NMDA injection. The
J Neurophysiol • VOL
locomotion improved by 17 days, which by then some extent
of locomotor recovery is likely to have occurred and probably
enhanced from the training received during experimentation.
The prolonged locomotor effects of NMDA was consistently
observed in all three intermediate-spinal cats (NG2, NG3,
NG5) as shown in Fig. 7. The step cycle duration recorded at
24 h after NMDA injection remain high as compared with the
predrug level. In two of three cats, the postdrug level was
significantly higher than the predrug level.
Latency of effect
While the three intermediate spinal cats markedly improved
locomotion after NMDA injection, the time it takes to reach
maximal locomotor performance (latency), however, differed
among them. In one cat (NG2, 7 days), marked locomotor
effects were apparent as early as 17 min after drug delivery; in
another cat (NG5, 6 days) while bouts of organized stepping
with foot placement and weight support were also observed at
2.5 h post-NMDA injection, these effects were extremely transient. In fact, maximal effects of NMDA were not seen until
the following day (24 h after injection) where a robust locomotor pattern (ⱕ0.6 – 0.7 m/s) with weight support and foot
placement was observed. Similar findings were seen in NG3 (7
days) where some locomotor effects could be observed at 1 h
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FIG. 3. NMDA elicited stepping movements in a 4-day spinal cat (CC7). A: raw EMG of hindlimb flexor (St, Srt) and extensor
muscles (VL, GL), duty cycles, and stick diagram of a 4-day spinal cat before NMDA injection. B: at 7 min post-NMDA injection,
(1 mM it), stepping movements was elicited and are characterized by some asymmetry, little weight support even with proper foot
placement on the left side. The cat was also unable to follow treadmill speed beyond 0.2 m/s.
post-NMDA (1 mM it) such as more regular alternating stepping movements and a more organized EMG pattern. However,
there was no foot placement during stepping until the following
To summarize, our results show that in the early postspinalization period (when there was no locomotor ability), NMDA
alone or with DHK at best increased some rhythmic stepping
but did not trigger locomotion with foot placement or weight
support in chronic spinal cats. In the intermediate postspinalization period, where the spinal cats began to show some
locomotor ability (very small steps with occasional foot/toes
placements), NMDA significantly enhanced the locomotor pattern with marked increase in step length, cycle duration, regular plantar foot placement and weight support, muscles activities and an organized EMG pattern. While the onset of the
aforementioned effects differed among the three spinal cats
tested, the duration of these effects were similar and were
mM it), while there was no changes in the locomotor pattern,
the threshold of electrical stimulation decreased to 375 ␮A
(40% of control). For the same stimulation intensity, the cutaneous reflex response was also bigger after NMDA injection as
shown in Fig. 8A. In this 3d-spinal cat (CC7), there was a small
LSrt response to electrical stimulation before NMDA injection.
After NMDA (1 mM it), with the same stimulating parameters,
there was a marked increase in LSrt, LSt, and RVL response.
In the intermediate phase postspinalization where NMDA (1
mM it) significantly improved the locomotor pattern, it did not
change the cutaneous reflex response (n ⫽ 2, 4 injections). For
example, the threshold of electrical stimulation at rest (lying on
the side) did not change after NMDA injection. With the same
stimulating intensity, the cutaneous reflex response was also
found to be similar as shown in Fig. 8B. It shows that in a
11-day spinal cat (NG2), the cutaneous reflex response, stimulated at same intensity (120 ␮A) was comparable before and
at 31 min post-NMDA (1 mM it).
Effects of intrathecal NMDA on cutaneous reflex excitability
In the early phase postspinalization, NMDA significantly
increased the cutaneous excitability in the spinal cats (n ⫽ 2).
In one spinal cat (CC7), the increase in cutaneous reflex was
also found to be dose-dependent. After the first NMDA injection (1 mM it), the threshold of electrical stimulation decreased
from 640 to 540 ␮A. After a subsequent NMDA injection (5
J Neurophysiol • VOL
Summary of results
First, during the early postspinalization period (ranging from
3– 6 days postspinalization), NMDA (1 mM it) did not trigger
robust locomotion (with weight support and foot placement) in
contrast to the noradrenergic agonist, clonidine. A higher dose
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FIG. 4. The effects of NMDA on locomotion during the intermediate phase (7 days postspinalization). A: stick diagram (1 step
cycle) and averaged EMG of hindlimb flexors (Srt, St) and extensors (GL, GM) synchronized to LSt during treadmill locomotion
(0.4 m/s) before any drug injection. A shaded bar was placed under the stick diagram representing the onset of the stance phase;
cat NG2. B: locomotion recorded at 17 min after NMDA (1 mM it). Note the marked increase in step cycle duration as well as the
marked increase in flexor and extensor muscle activities. C: locomotion at 24 h after NMDA injection. Note the decrease in the
muscle activities in RSrt and RVL. D: locomotion at 72 h after NMDA injection, a further deterioration in the muscle activities
was seen.
were capable of initiating some rudimentary stepping, NMDA
markedly enhanced the preexisting rudimentary locomotor pattern in all three cats with preferential enhancement of the flexor
muscle activity. Third, in contrast to a ␣2 noradrenergic agonist, which markedly decreased the cutaneous excitability
(Chau et al. 1998a,b), NMDA dose-dependently increased the
cutaneous excitability. Fourth, NMDA exerted a prolonged
effect on locomotor activities (⬎24 h).
Initiation of locomotion during the early phase
(5 mM it) of NMDA triggered spontaneous fast paw shaking
without effects on locomotion. Second, in the intermediate
phase, ⬃7– 8 days postspinalization, when the cats (n ⫽ 3)
1. Burst duration in ms of flexor and extensor muscles during locomotion in intact and in spinal cat pre and post-NMDA (1 mM it)
7 d spinal
cat NG2
(0.4 m/s)
86 ⫾ 18
76 ⫾ 18
526 ⫾ 65
472 ⫾ 76
17 min post-NMDA
(1 mM)
24 h postNMDA
72 h postNMDA
293 ⫾ 56 (340 of intact)
267 ⫾ 88 (351)
569 ⫾ 64 (108)
632 ⫾ 31 (133)
290 ⫾ 52 (337)
208 ⫾ 43 (273)
407 ⫾ 72 (77)
457 ⫾ 58 (96)
216 ⫾ 54 (251)
342 ⫾ 40 (450)
445 ⫾ 81 (84)
363 ⫾ 49 (76)
8 d spinal
cat NG3
(0.4 m/s)
1 h post-NMDA
(1 mM)
20 h postNMDA
48 h postNMDA
170 ⫾ 30
577 ⫾ 64
536 ⫾ 49
684 ⫾ 58
283 ⫾ 89 (148)
163 ⫾ 44 (26)
143 ⫾ 38 (84)
322 ⫾ 54 (56)
340 ⫾ 51 (63)
329 ⫾ 44 (48)
259 ⫾ 83 (152)
346 ⫾ 131 (59)
497 ⫾ 132 (92)
345 ⫾ 59 (50)
Values are means ⫾ SD. Parentheses enclose percentages that were calculated as a percentage of the intact locomotion. Results from cat NG2 are also used
in Fig. 6 and part of Fig. 7. LSt and RSt, left and right semitendinosus; LGL and RGL, left and right gastrocnemius lateralis; LVL and RVL, left and right vastus
lateralis; n/a, not available.
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FIG. 5. Changes in EMG activities and cycle duration in an intermediate
spinal cat (NG2) after NMDA injection (1 mM it). A: histogram of flexor and
extensor burst duration during intact state and at different times after NMDA
injection. Note that the burst duration of St was increased 2- to 3-fold as
compared with during the intact locomotion. B: changes in the averaged ankle
extensor burst (LGL) duration and step cycle duration as a function of treadmill speed at 24 h after NMDA injection. The number in parentheses indicate
the number of cycles measured.
Previous work from our laboratories have shown that ␣2
noradrenergic agonists (clonidine, oxymetazoline, and tizanidine) triggered coordinated locomotion with weight support in
chronic spinal cats within the first week postspinalization (Barbeau et al. 1987; Chau et al. 1998a,b). In light of the important
role of NMDA in locomotion and the success of NMDA in
triggering locomotion in a variety of preparations (see introduction), it is surprising that we were unable to initiate robust
locomotion in adult cats early on after spinalization as did the
noradrenergic agonists. It is possible that in an awake behaving
animal, a bolus injection of NMDA may activate receptors that
are not involved in the generation of locomotion, such as pain
receptors (Raigorodsky and Urca 1987). The increase in cutaneous excitability and the spontaneous “fast paw shake” (FPS)
movements that we observed (with higher dose of NMDA) is
also likely the result of excitation of sensory pathways. That is,
the NMDA receptors are diffusely distributed in the CNS, the
widespread activation of NMDA receptors in awake animal
might lead to interference with the expression of locomotion.
This is in accordance with recent studies that while NMDA
triggered highly regular and long-lasting locomotor rhythm in
the isolated turtle spinal cord-hindlimb preparation (in vitro),
NMDA was far less effective in the immobilized spinal turtle
preparation (in vivo) where it triggered only weak and irregular
bursting from hip nerves or no knee extensor discharges (Currie 1999). The relative inefficiency of NMDA in in vivo turtle
preparation but not in vitro preparation supports the notion that
excessive sensory stimulation (by NMDA) in the in vivo state
may have interfered with the expression of locomotor rhythm.
A higher dose of NMDA (ⱕ10 mM it) did not elicit any
locomotor activities but spontaneous hindlimb movements resembling FPS movements (Smith et al. 1980). FPS is characterized by high-frequency muscles activation (alternating
flexor and extensor) with short cycle duration (55–110 ms)
(Smith et al. 1980). Our findings show that the frequency of the
FPS rhythmic motor pattern in chronic spinal cats elicited by
NMDA is 8 –10 Hz, which is out of the range for locomotion
and is similar to the FPS previously recorded in fictive cat
preparation (Pearson and Rossignol 1991). In the study by
Douglas et al. (1993), they also reported that during fictive
locomotion evoked by NMDA, high-frequency rhythmic activity (7.5 Hz) was observed and occasionally interrupted the
In addition to paw shake response, EAA have also been
reported to produce other types of motor rhythm such as
spontaneous wiping, hindlimb kicking, and jumping movements in adult spinal frogs (McClellan and Farel 1985).
Thus it appears that a mere increase in NMDA concentration
was not sufficient to initiate locomotion. Indeed, it is known
that there is a posttraumatic release of EAA that acts on
NMDA receptors contributing to the secondary damage
(Haghighi et al. 1996; Young 1988) after acute spinal cord
injury, and as such this does not appear to trigger locomotion.
Role of NMDA on mediating cutaneous reflex
An NMDA-antagonist, AP5, when applied to the hindlimb
enlargement of the turtle spinal cord, was found to reduce the
response amplitude of the flexion reflex (Stein and Schild
1989). This evidence suggests that NMDA receptors on sen-
FIG. 7. Effects of NMDA in 3 intermediate spinal
cats at 24 h after injection. Histogram showing the cycle
duration in 3 intermediate spinal cats ranging from 6 to
8 days postspinalization. For each cat, the step cycle
duration was obtained during intact state (䊐), pre-NMDA
injection (1), and at 24 h after NMDA injection (■).
*, significant changes compared with the pre-NMDA
values (P ⫽ 0.05).
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FIG. 6. The prolonged effects of NMDA on locomotion of an intermediate spinal cat NG2. The step cycle duration during
treadmill locomotion (0.4 m/s) shown as a function of time postspinalization after NMDA injection (2) in 1 spinal cat. {, cycle
duration during spinal locomotion before NMDA injection; }, cycle duration after NMDA injection. For example, on 7 days
postspinalization, cycle duration before and 17 min after NMDA was shown by { and }, respectively. The cycle duration during
intact locomotion (means ⫾ SD) was shown by 1.
sory interneuron play a role in the spinal cord processing of
cutaneous information.
Similar to our findings, intrathecal NMDA (1 mM) was also
found to increase persistent hindlimb flexion (induced by electrical stimulation) in anesthetized intact and spinal rat (3 days),
and pretreatment of MK-801 prevented the enhancement of
flexion (Moore et al. 1992). The spontaneous FPS we observed
after a high dose of NMDA injection (⬎5 mM it) may be
related to an increased excitability of the cutaneous afferent
innervating the paw (Smith et al. 1980).
Effects of NMDA in the intermediate phase postspinalization
At ⬃6 – 8 days postspinalization, the cat was able to make a
few rudimentary steps that may be related to a decrease in
endogenous release of EAA or perhaps reflects that some
degree of functional neuronal connectivity required for stepJ Neurophysiol • VOL
ping was reestablished. Perhaps once this connectivity is in
place, NMDA enhanced the excitation of the CPG through
mechanisms implicating motoneurons and interneurons.
The injection of NMDA may also mimick the effect of
primary afferent stimulation. Electrophysiological recordings
of feline spinal neurons showed that L-glutamate was released
by terminals of low-threshold primary afferent on stimulation
that acts primarily on quisqualate receptor, and the excitatory
interneuron, in response to the stimulation, releases L-aspartate,
which acts on the NMDA receptors (Davies and Watkins
1983). Thus L-glutamate and L-aspartate were suggested to be
the transmitter involved in mono- and polysynaptic responses,
respectively. This notion was supported in another study where
recording were made from the spinal dorsal horn neurons in the
isolated neonatal rat hemisected spinal cord preparation. It was
also found that the excitatory amino acids released at the
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FIG. 8. The cutaneous reflex response during the early and
intermediate phase postspinalization. A: averaged responses to
electrical stimulation of superficial peroneal nerve while standing in a 3 days spinal cat (CC7) before (13 stimulations) and 30
min after NMDA (1 mM it) injection (25 stimulations). The
current of stimulation was 640 ␮A before and after NMDA
injection. B: averaged responses to electrical stimulation of superficial peroneal nerve while lying on the right side in a 11-day
spinal cat (NG2) before and 35 min after NMDA (1 mM it)
injection. The current of stimulation was 120 ␮A before and
after NMDA injection.
Intrinsic neuronal mechanisms
NMDA receptors have been shown to play a key role in
affecting the intrinsic neuronal properties of the CPG. For
example, NMDA receptors have been implicated in the rhythmic bursting activity of spinal neurons during fictive locomotion (Grillner and Wallen 1985; Wallen and Grillner 1987).
NMDA-induced intrinsic motoneuronal membrane potential
oscillations (TTX-resistant) was also reported in tadpole (Sillar
and Simmers 1994) and neonatal rat (Hochman et al. 1994;
McClean et al. 1997). These NMDA-induced oscillatory
changes in membrane potential may play a role in mediating
various rhythmic motor pattern such as locomotion or perhaps
the FPS (McClean et al. 1997). NMDA-induced intrinsic membrane properties were also found in interneurons (Grillner and
Wallen 1985; Sigvardt et al. 1985; Wallen and Grillner 1987).
The voltage-dependent properties of NMDA receptor-ion
channel appears to play a key role in the generation of lowfrequency, steady burst pattern (Brodin and Grillner 1985a,b;
Brodin et al. 1985). NMDA, along with other substance such as
serotonin, noradrenaline, has also been found to induce plateau
potentials in the motoneurons and interneurons in the spinal
cord (see Kiehn et al. 1997).
Plateau potentials are intrinsic properties of the neurons that
maintain a prolonged state of depolarization in the absence of
synaptic inputs, thus significantly amplifying the level of excitatory output (Kiehn 1991). Plateau potentials have been
reported in the motoneurons of spinal cats during fictive locomotion induced by L-DOPA, clonidine, or a serotoninergic
precursor, 5-hydroxytryptophan (5-HTP) (Conway et al. 1988;
Hounsgaard et al. 1988; Schomburg and Steffens 1996), and
interneuron of spinal rats (Kiehn et al. 1996) induced by
NMDA and 5-HTP. The main role of the NMDA-induced
intrinsic membrane properties (both motoneurons and interneurons) and plateau potential is to shape the duration and enhance
the amplitude of the final motoneuronal output (Kiehn 1991)
thereby modifying the characteristics of the induced motor
pattern (Kiehn et al. 2000). NMDA-induced membrane oscillation may not be needed for rhythm generation as transmitterinduced locomotor activity could still be possible in tadpole,
lamprey, and neonatal rats in Mg2⫹-free saline, which abolishes the NMDA-induced membrane oscillations but not the
EAA-mediated excitatory postsynaptic potentials (EPSPs) (for
J Neurophysiol • VOL
review, see Kiehn et al. 1997). It is therefore suggested that the
role of NMDA in regulating pattern generation by the spinal
CPG may be through amplification and enhancement of the
synaptic inputs between the CPG elements or transmission
from sensory pathways (Kiehn et al. 1997).
NMDA-mediated prolonged effects
Prolonged effects (24 –72 h) on locomotion exerted by a
single dose of NMDA was observed during the intermediate
phase of locomotion. How could these prolonged effects be
explained from other studies? The actions of spinal NMDA
receptors was extensively studied in the pain control system
where NMDA receptors have also been reported to have prolonged effects as documented from behaviors in animal models. The plasticity of pain control system was proposed to
involve mechanism similar to those involved in learning
(Svendsen et al. 1998). Both NMDA and AMPA receptors
have been shown to contribute to the long-term potentiation
(LTP) of the C fibers after tetanic stimulation of sciatic nerve,
lasting for ⱖ6 h. In another study, while activation of NMDA
receptors (superficial infusion) was shown to induce LTP of the
spinal C-fiber-evoked potentials in spinalized rats, it failed to
induce LTP in intact rats, suggesting a critical role by the tonic
descending inhibition.
Potentiation of neuronal NMDA responses by substance P is
thought to play a role in nociception and the persistent pain
(Rusin et al. 1993). It has also been suggested that NMDA
receptor activation releases substance P from nociceptive terminals (Liu et al. 1997). Recent study reports a co-localization
of NMDA receptors and substance P receptors in rat spinal
cord at all levels (Benoliel et al. 2000).
It has been shown in the lamprey that bath application of
tachykinin substance P for 10 min resulted in a long-lasting
(⬎24 h) modulation of the NMDA-evoked locomotor network
activity (Parker and Grillner 1998). Tachykinin acts on sensory
inputs in the lamprey, modulating excitability of the primary
afferent and sensory interneuron (Parker and Grillner 1996).
The tachykinin-mediated long-lasting effects (⬎2 h) was found
to involve protein synthesis (Parker and Grillner 1999; Parker
et al. 1998).
Thus the long-term effects exerted by NMDA receptors
activation in our study may involve the aforementioned mechanisms, such as induction of LTP as seen in pain and reflex
studies. Also, given the close interactions between NMDA
receptors and neuropeptides such as tachykinin, we cannot
exclude the involvement of neuropeptides (e.g., substance P),
which may cascade a sequence of cellular changes contributing
to the long-term effects on locomotor network activity.
We acknowledge the expert technical help of J. Provencher during surgeries,
experiments, data analyses, and figures preparation. We also acknowledge F.
Lebel, P. Drapeau, G. Messier, C. Gagner, and J. Faubert for technical
assistance. Many thanks to Dr. T. Reader for helpful discussions.
C. Chau was supported by the Fonds Pour la Formation de Chercheurs et
l’Aide à la Recherche (FCAR), the Canadian Neuroscience Network, the
network of centers of excellence and the Groupe de Recherche sur le Système
Nerveux Central. H. Barbeau. is a scholar of the Fonds de la Recherche en
Santé du Québec. L. Jordan was supported by a Canadian Institute for Health
Research (CIHR) professorship. This work was supported by a group grant
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myelinated primary afferent terminal on stimulation act on
kainate/quisqualate receptors (which was responsible for the
short-latency response) and that coactivation of NMDA receptors occurs at a slower time course (the long latency response)
(Morris 1989). Therefore in our study, the marked locomotor
effect of NMDA observed in the intermediate spinal cats may
indeed be partly attributed to the NMDA activation of sensory
pathway resembling that of peripheral stimulation. This may
partly explain the marked increased in the flexor burst activity
observed. It may also be related to the perineal stimulation
given during locomotion in early or intermediate spinal cats,
which often produced a somewhat exaggerated hindlimb flexion as shown in the stick diagrams in Fig. 4B. In fact, differential modulation of EMG burst duration and amplitude of
flexors and extensors in chronic spinal cats has already been
documented using various drugs (Barbeau and Rossignol
from the CIHR, the Canadian Neuroscience Network, and partly by an FCAR
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