Centre de Recherche en Sciences Neurologiques, Département de Physiologie, Faculté de Médecine, Université de Montréal, Montreal, Quebec H3C 3J7, Canada
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ABSTRACT |
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Giroux, Nathalie,
Tomás A. Reader, and
Serge Rossignol.
Comparison of the Effect of Intrathecal Administration of
Clonidine and Yohimbine on the Locomotion of Intact and
Spinal Cats.
J. Neurophysiol. 85: 2516-2536, 2001.
Several
studies have shown that noradrenergic mechanisms are important for
locomotion. For instance, L-dihydroxyphenylalanine (L-DOPA) can
initiate "fictive" locomotion in immobilized acutely spinalized
cats and 2-noradrenergic agonists, such as
2,6,-dichloro-N-2-imidazolidinylid-enebenzenamine (clonidine), can induce treadmill locomotion soon after spinalization. However, the activation of noradrenergic receptors may be not essential
for the basic locomotor rhythmicity because chronic spinal cats can
walk with the hindlimbs on a treadmill in the absence of noradrenergic
stimulation because the descending pathways are completely severed.
This suggests that locomotion, in intact and spinal conditions, is
probably expressed and controlled through different neurotransmitter
mechanisms. To test this hypothesis, we compared the effect of the
2 agonist, clonidine, and the antagonist (16
, 17
)-17-hydroxy yohimbine-16-carboxylic acid methyl ester hydrochloride (yohimbine), injected intrathecally at
L3-L4
before and after spinalization in the same cats chronically implanted with electrodes to record electromyograms (EMGs). In intact cats, clonidine (50-150 µg/100 µl) modulated the locomotor pattern
slightly causing a decrease in duration of the step cycle accompanied
with some variation of EMG burst amplitude and duration. In the spinal state, clonidine could trigger robust and sustained hind limb locomotion in the first week after the spinalization at a time when the
cats were paraplegic. Later, after the spontaneous recovery of a stable
locomotor pattern, clonidine prolonged the cycle duration, increased
the amplitude and duration of flexor and extensor bursts, and augmented
the foot drag at the onset of swing. In intact cats, yohimbine at high
doses (800-1600 µg/100 µl) caused major walking difficulties
characterized by asymmetric stepping, stumbling with poor lateral
stability, and, at smaller doses (400 µg/100 µl), only had slight
effects such as abduction of one of the hindlimbs and the turning of
the hindquarters to one side. After spinalization, yohimbine had no
effect even at the largest doses. These results indicate that, in the
intact state, noradrenergic mechanisms probably play an important role
in the control of locomotion since blocking the receptors results in a
marked disruption of walking. In the spinal state, although the
receptors are still present and functional since they can be activated
by clonidine, they are seemingly not critical for the spontaneous
expression of spinal locomotion since their blockade by yohimbine does
not impair spinal locomotion. It is postulated therefore that the
expression of spinal locomotion must depend on the activation of other
types of receptors, probably related to excitatory amino acids.
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INTRODUCTION |
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Lundberg and coworkers
(Jankowska et al. 1967a,b
) have shown that the
intravenous (iv) administration of L-dihydroxyphenylalanine (L-DOPA), a
precursor of noradrenaline, together with the monoamine oxidase
inhibitor nialamide, in acutely spinalized and paralyzed cats
profoundly modified spinal circuits. Indeed, long-latency and -duration
discharges, often rhythmically organized in antagonist muscle nerves,
were evoked by stimulation of sensory nerves. The same drugs were also
found to induce a detailed pattern of locomotion in paralyzed cats,
indicating the existence in the cat spinal cord of a central pattern
generator (Grillner and Zangger 1979
). In acutely
spinalized adult cats, Forssberg and Grillner (1973)
showed that
2,6,-dichloro-N-2-imidazolidinylidenebenzenamine
(clonidine), an
2-noradrenergic agonist
injected iv, could evoke hindlimb locomotion on a treadmill. In chronic
spinal cats (T13), clonidine and other
2-noradrenergic agonists such as tizanidine
and oxymetazoline injected intraperitoneally or intrathecally have been
shown (Barbeau et al. 1987
; Chau et al.
1998a
) to initiate locomotion within the first week following
spinalization. In late spinal cats capable of walking with the
hindlimbs without drugs, these
2 agonists exert a potent modulation of the locomotor pattern. For example, clonidine increases the cycle duration and flexor muscle burst duration
as well as decrease the extensor activity and consequently the weight support.
The major source of spinal noradrenaline (NA) afferents are cell groups
in the brain stem, namely the locus coeruleus and subcoeruleus, the
nucleus of Kölliker-Fuse as well as the medial and lateral
parabrachial nuclei (Dahlström and Fuxe 1964a,b
). After a complete spinal transection, all descending pathways, including
NA fibers, are destroyed. Nevertheless adult cats usually recover
within a few weeks the ability to walk with the hindlimbs on the
treadmill (for a review, see Rossignol 1996
;
Rossignol et al. 1999
). These results indicate then that
in the adult spinal cat, since the descending monoaminergic systems are
absent, the stimulation of noradrenergic receptors is not essential for
triggering or organizing the basic locomotor pattern. Therefore
locomotion in the spinal state may depend on other transmitter systems
still present after the spinal transection. However, this does not
prevent the stimulation of these receptors from affecting locomotion in the spinal state as mentioned in the preceding text nor does it negate
that in intact cats, NA receptor stimulation may be essential for the
normal modulation of the locomotor pattern.
The aim of the present study was thus to investigate the role of the
noradrenergic system on locomotion by injecting intrathecally (i.t.)
the 2-noradrenergic agonist clonidine and the
2-noradrenergic antagonist (16
,
17
)-17-hydroxy yohimbine-16-carboxylic acid methyl ester
hydrochloride (yohimbine at lumbar segments
(L2-L4) in the same cats,
before and after a complete spinal cord transection at
T13. It is indeed important to know the effects
of drugs in the intact and spinal states since the state of the
receptors change after spinalization (Giroux et al.
1999b
). Furthermore as spinalization eliminates presynaptic
receptors by removing all descending noradrenergic terminals, receptors
activated in the spinal state are located postsynaptically that allows
discrimination between pre- versus postsynaptic effects of drugs.
Preliminary results have been published in abstract form (Giroux
et al. 1996
, 1998
, 1999a
).
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METHODS |
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General protocol
Adult cats (n = 3) were trained during a 3- to
4-wk period to walk at different speeds (0.2-0.8 m/s) on a
motor-driven treadmill. When the cats had been trained to walk at a
constant speed for ~15-20 min, they were implanted with chronic
electromyographic (EMG) electrodes in muscles of the hindlimbs
(Bélanger et al. 1996) as well as with an i.t.
cannula. Nerve-cuff electrodes were placed on the superficial peroneal
nerve just above the ankle of both sides to test reflexes. After
obtaining baseline values for locomotion in the intact state,
drug-injection experiments were performed while in the intact state.
The cats were then spinalized at T13 under
general anesthesia and trained for 3-4 wk to walk on the treadmill.
When the cats recovered spinal locomotion, the same drugs were
re-injected to allow a comparison of the effects of the same drugs in
the intact and the spinal states. Overall these experiments lasted for
periods of 6 mo to 2 yr.
Implantations
All surgical procedures were performed in aseptic conditions and approved by the Comité de Déontologie pour l'Expérimentation Animale from the l'Université de Montréal. The cats were premedicated with acepromazine maleate (Atravet, 0.1 mg/kg), ketamine (10 mg/kg), and glycopyrrolate (0.01 mg/kg) injected subcutaneously and anesthetized with 1-3% isofluorane. Lactate Ringer solution was administered during the surgery through an intravenous catheter. The body temperature was monitored with a rectal thermometer and constantly adjusted by a heating pad.
The i.t. cannulation procedure was adapted from Espey and Downie
(1995) and already described in detail (Chau et al.
1998a
). Briefly, a Teflon tubing (24W) connected to an adaptor
(cannula pedestal and dustcap) was fixed to the skull with dental
acrylic cement and constituted the cannula inlet. The other extremity of the tubing was inserted into the subarachnoid space through an
opening made in the atlanto-occipital ligament and pushed down to
L3-L4 as measured
externally by counting spinous processes. The cannula was flushed three
to four times per week with a bolus of 100 µl of saline solution
(0.9%) to prevent blocking. X-rays performed in one cat
(NG2) showed the distribution of a radio-opaque substance,
iohexol (Omnipaque, Amersham Canada, Ontario, CA), injected through the
i.t. cannula. The tip of the cannula was located dorsally between
L3 and L4 segments, and the
radio-opaque substance diffusion remained within the lumbar
L3-L5 level. This observation was also confirmed postmortem, in the same cat,
with a bolus injection of 200 µl of Fast Green dye (see Fig.
1A). The drug diffusion was
limited by the formation of a spinal fibrosis pocket at the distal
extremity of the cannula. The location of the cannula's tips for the
other cats NG3 and NG5 are shown in Fig. 1,
C and D. The cannula of the cat NG3
terminated dorsally on the left side at L2
segment and for the cat NG5 the end of the cannula was
located dorsolaterally on the right side, just below the ventral root,
at L4.
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After implanting chronic EMG electrodes (Bélanger et al.
1996; Chau et al. 1998b
), two 15-pin head
connectors (TRW Electronic Components Group) previously attached to 14 pairs of Teflon-insulated stainless steel wires for each connectors
were fixed to the skull with acrylic cement. Each pair of wires was
inserted subcutaneously up to a small skin incision made over the hind
limb muscles selected in this study. Then, 2-3 mm of the wire was
scraped off to remove the insulation and inserted within the muscle.
One wire for each connector was placed under the skin, and it served as
an electrical ground.
Bipolar nerve-cuff electrodes were implanted and used to stimulate the
superficial peroneal nerve in both hindlimbs (Julien and
Rossignol 1982). Two pairs of Teflon-insulated stainless steel wires were attached with two 2-pin head connectors and fixed to the
skull. Pairs of wires were inserted subcutaneously (~6 mm between
electrodes leads) to a nerve cuff made from polymer (Culk Dentsply
International). The superficial peroneal nerve was placed in the cuff
in contact with the wires, and the cuff was completely sealed off with
the polymer.
Spinal cord transection
At the end of the experiments in the intact period, the cats were anesthetized, and a laminectomy was performed at the T13 vertebra. The dura was carefully removed, and 2% xylocaïne was first applied on the surface of the cord and then injected into the spinal cord. After having localized the i.t. cannula, the spinal cord was completely transected using micro scissors. The space between the rostral and caudal ends was solidly packed with a sterile absorbable hemostat (Surgicel, oxidized regenerated cellulose).
Postoperative cares
After surgery, the cats were placed in an incubator until they regained consciousness and then returned to their individual cages with food and water ad libitum. During the first two and three postoperative days, cats received 0.005-0.01 mg/kg sc of Buprenorphine (every 6 h) for analgesia. The cage floors were covered with a foam mattress to reduce discomfort and skin ulceration. Cats were attended daily for general inspection, cleaning the head connectors and hindquarters, manual bladder expression, and flushing the cannula when appropriate.
Drug injections
The 2-noradrenergic agonist
2,6,-dichloro-N-2-imidazolidinylid-enebenzenamine
(clonidine) from Sigma and the antagonist (16
, 17
)-17-hydroxy
yohimbine-16-carboxylic acid methyl ester hydrochloride (yohimbine)
from RBI were used in this study. The drugs were injected as a bolus of
100 µl into the subarachnoid space of the spinal cord through the
inlet of the cannula, and a subsequent bolus injection of saline (100 µl) was made to flush the drug outside the cannula, the dead space of
the cannula being about 100 µl. Yohimbine was dissolved in sterile
distilled water and given at a concentration of 5.1-40.9 mM while
clonidine was dissolved in saline solution (0.9%) and administered at
a dose of 1.9-5.6 mM. These values were based on previous studies with
these drugs in chronic spinal cats (Brustein and Rossignol
1999
; Chau et al. 1998a
).
Recording and analysis procedures
After the implantation and before any drug injections, recordings of locomotion were done in the intact state as the cat walked freely at different speeds (0.2-0.8 m/s) and tilts (15° up or 15° down) on a treadmill belt. The cats were also trained to walk on a horizontal ladder with eight round rungs (3 cm in diameter) spaced by ~20 cm. The later task was recorded only on video tape and studied only in the intact state. These recording served as baseline controls (intact trials). During each drug-injection trial, similar recording were done before (predrug trial) and at different times after the drug injection (postdrug trial).
The experiments with the spinal cats were made when they had recovered a well-coordinated locomotor pattern of the hindlimbs with full weight support of the hindquarters and plantar foot placement. For spinal locomotion, the forelimbs were placed on a platform situated 2 cm above the treadmill, while the hindlimbs walked on the belt and the tail was held to maintain equilibrium of the hindquarters. A Plexiglas separator was place between the hindlimbs to prevent crossing of the hindlimbs.
The EMG signals were differentially amplified (bandwidth of 100 Hz to 3 kHz) and recorded on a 14-channel tape recorder (Vetter Digital, model 4000A PCM recording adapter) with a frequency response of 1.2 kHz per channel. The EMG recordings were synchronized to the video images of the hindlimbs using a digital SMPTE (Society for Motion Picture and Television Engineers) time code. This time code was recorded simultaneously on the EMG tape, the audio channel of the VHF tape as well as into the video image.
Video images of the side view of the left hind limb during locomotion were captured using a digital camera (Panasonic 5100, shutter speed 1/1000 s) and recorded on a video cassette recorder (Panasonic, AG 7300). Reflective markers were glued to the skin over the bony landmarks (iliac crest, femoral head, knee joint, lateral malleolus, metatarso-phalangeal or MTP joint and the tip of the 4th toe) of the left (ipsilateral) hind limb. Two additional markers placed on the trunk of the animal served for calibration (10 cm) to reduce the parallax error.
The kinematic analyses were carried out using a two-dimensional Peak Performance system (Peak Performance Technologies, Englewood, CO). The video images were digitized and the x-y coordinates of different joint markers were obtained at a frequency of 60 fields/s. These coordinates were used to calculate angular joint movements and could be displayed as continuous angular displacements or stick diagrams of one step cycle.
Reflex testing
ELECTRICAL STIMULATION. The superficial peroneal nerve was stimulated by a single pulse of 250 µs at 0.45 Hz (Grass S88 stimulator, Quincy, MA) through the nerve-cuff electrodes. The stimulation was delivered at rest when the cat was laying down on the treadmill. Selected EMGs were displayed on an oscilloscope (Tektronix 2214) and the threshold of stimulation was set at the current value required to evoke a small short-latency (10 ms) response in the semitendinosus muscle (St) in 50% of the trials.
FAST PAW SHAKE. Fast paw shake was elicited by holding the spinal cat in the air and then dipping the paw into a bowl of warm water. During the fast paw shake, EMG signals and video images were recorded, but only the EMG signals were analyzed.
Histology
At the end of the experimental series, the animals were killed with an overdose of pentobarbital sodium, and the spinal cord were removed and divided into 4- to 6-cm-thick blocks that were rapidly frozen for autoradiographic analysis in other studies. To assure the completeness of the spinal transection, the segment of the encompassing lesion was completely removed and cut in sagittal 10-µm-thick sections that were stained with the Klüver-Barrera method for histological observations.
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RESULTS |
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These results were obtained from three adult cats in both the intact condition and after a complete spinal transection. In each condition, drugs were injected several times on different days and at different concentrations. The schedule and dosages of clonidine and yohimbine injection during control and spinal states are show in Fig. 2. Cat NG2 was kept 658 days in the intact state and 158 days after the spinal transection while both NG3 and NG5 were kept 263 and 127 days as intact and 114 and 70 days after the spinalization, respectively. The results reported in this study are from selected experiments in different cats in both conditions. Even if some experiments were not analyzed in detail, the videotapes and EMG data were always reviewed to verify the similarities or differences in the effect of drugs. In the spinal condition, except for one experiment with clonidine (Fig. 6), all drugs were administered when the cat had recovered a well-organized locomotor pattern with adequate foot placement and weight support.
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Effects of clonidine on locomotion
INTACT CAT.
Level walking.
In this study, high doses of clonidine correspond to 100-150 µg/100
µl and are well known to have important locomotor effects in spinal
cats (Chau et al. 1998a). Since clonidine induced in the
intact cats side effects, i.e., nausea and drowsiness, lower doses (50 µg/100 µl) were used in a few trials.
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SPINAL CAT.
Level walking: initiation in early spinal cat.
The ability of clonidine to trigger locomotion in complete spinal cats
has already been well documented (Barbeau et al. 1987; Chau et al. 1998a
), but the comparison of the effects of
clonidine in the same cat before and at various times after
spinalization has not been made. In Fig.
6, three days after the spinalization, the same cat as shown in Fig. 3 in the intact state is now paraplegic. A strong perineal stimulation could sometimes produce episodes of faint
stepping on the treadmill (Fig. 6A), but this locomotor activity was not well organized as shown by the duty cycle of the
left/right limbs and EMGs traces (Fig. 6, D and
C) and was so small that the hip, knee, ankle, and MTP
joints barely moved (Fig. 6B). The paws were always
dragged on the treadmill belt, and no plantar foot placement nor weight
support were seen before clonidine injection. As illustrated in Fig. 6,
E-H, one bolus injection of clonidine (100 µg/100
µl) triggered an adequate locomotor pattern with good bilateral
placement of the feet on the plantar surface and weight support of the
hindquarters, requiring only light perineal stimulation. When compared
with predrug conditions, there was a marked increase in the step length
as shown in the stick diagram (Fig. 6E) as well as by
the increase in the total angular excursion at the hip, knee, ankle,
and MTP joints (Fig. 6F). This spinal locomotion was
stable, adaptable to speed, and sustained, i.e., the animal could walk
for a long period of time (10-15 min), at different treadmill speeds
(0.1-1 m/s), and with a regular locomotor pattern.
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Effects of clonidine on cutaneous reflex excitability
After clonidine administration (50-150 µg/100 µl), the amplitude of the reflex response evoked by electrical stimulation of the superficial peroneal nerve decreased in both intact (10/12 injections) and spinal cats (4/5 injections); these effects are illustrated for cat NG2 in Fig. 8, A and B. In the intact cat (Fig. 8A), despite a much stronger stimulating current of 600 µA (3 times predrug threshold), there was still a marked decrease in amplitude of the short latency response in the St muscle. Similarly, in a spinal cat at 158 days (Fig. 8B), the same stimulation used before clonidine abolished the response in Srt and tibialis anterior (TA) muscles and markedly decreased the response in the St muscle.
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The time course of the effect on the reflex excitability was analyzed
in two intact cats (NG2 and NG3) and is shown in
Fig. 9, A and B.
This time course was evaluated by measuring the stimulation threshold
at rest at different time periods following clonidine administration.
The threshold of the stimulation was defined by observing a just
detectable response in the St muscle at rest. After clonidine, the
threshold increased and this persisted for 30-60 min before returning
to control values by 120-180 min. During the 30- to 60-min
postinjection, the cat was drowsy and unwilling to walk at high
treadmill speeds. The return of reflex excitability coincides with the
return of the ability to cope with high treadmill speeds as observed
during the predrug session and with the disappearance of side effects;
this time course of clonidine effects seems to be shorter in duration
for intact (2-3 h) than for late spinal cats (6 h) (Chau et al.
1998a).
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As previously shown in other studies (Barbeau et al.
1987; Chau et al. 1998a
), clonidine abolished
the fast paw shake response in 4/4 experiments. Figure 8C
shows an example of the fast paw shake response before and after
clonidine in cat NG5. In a 41-day postspinalization cat, 30 min after clonidine, the fast paw shake response was completely
abolished by a small dose of clonidine (50 µg/100 µl).
In summary, clonidine slightly modulated the locomotor pattern in intact cats. High doses (100-150 µg/100 µl) only caused a decrease in duration of the step cycle and, in some muscles, slight variations in burst amplitude and duration as well as difficulty to follow treadmill speeds higher than 0.4 m/s. Lower doses caused only some minor variations in muscle amplitude and duration. Also, after clonidine all cats were able to walk either on 15° slopes (uphill or downhill) or on a horizontal ladder. On the other hand, in the same cats but in the spinal state, clonidine triggered robust and sustained hind limb locomotion in the first week after the spinalization at a time when cats were paralyzed. Later when the cats had recovered a stable spontaneous locomotor pattern, clonidine prolonged the cycle duration, increased the amplitude and duration of flexor and extensor muscles, and augmented the foot drag at the onset of swing. Furthermore, in both intact and spinal conditions, the excitability of the cutaneous reflexes decreased significantly after clonidine administration.
Effects of yohimbine on locomotion
INTACT CAT. Level walking. The overall effects of yohimbine administration in intact cats NG3 and NG5 are illustrated in Figs. 10 and 11, respectively. Four minutes after a bolus injection of yohimbine (800 µg/100 µl), the cat had walking deficits as shown by the tracing of the hind limb position taken from the video recording (Fig. 10B). There was a clear asymmetrical posture of the hindquarters and an asymmetry of stepping between the two hindlimbs leading to the turning of the hindquarters to one side. There was an abduction of the left hindpaw particularly pronounced at the end of stance and an adduction of the right hindpaw. The side of the turning was different for each cat and was not obviously related to the side of the cannula's tip as determined at the autopsy. Cats NG3 and NG5 turned on the right side (abduction of the left hindpaw), while NG2 turned on the left side (abduction of the right hindpaw). However, in some experiments, the effect could start on one side and continue on the other side.
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SPINAL CAT. The ability of yohimbine (400-1,600 µg/100 µl) to modulate the spontaneous locomotion was assessed in the late spinal state, at a time when cats showed a stable locomotor pattern. As shown in Fig. 15, A-D, the same cat illustrated in Fig. 11 but now after 32 days postspinalization, the locomotor pattern was now well established and consisted of a well organized EMG activity with full weight support and correct foot placement. Ten minutes after a bolus injection of yohimbine (1,600 µg/100 µl), there was no significant change in the locomotor pattern when compared with control as shown by the stick diagram, the angular displacement of all joints, the duty cycle and the EMG activity (Fig. 15, E-H). The amplitude, the duration and pattern of the EMG did not change significantly after yohimbine. Furthermore, in contrast to the intact cat, yohimbine caused no stepping irregularities. As shown in Fig. 13, C and D, the same dose of yohimbine (1,600 µg/100 µl) that produced a pronounced increase in the variability in the consecutive step cycle duration and in the interlimb coupling in the intact state (Fig. 13, A and B) caused no significant changes in step or coupling variability in the same cat after the spinalization. This was reflected by the same pre- and postfluctuation in the consecutive cycle duration (Fig. 13C) and interlimb coupling between the right and left lift (Fig. 13D). These results were always seen in all spinal cats and are presented in Table 2. In all trials with spinal cats, there were no significant differences in the coefficient of variation of the step cycle duration between pre (7-12%)- and post (6-10%)-yohimbine administration. Similarly, no differences were found in the CV of the mean interlimb coupling (pre = 8-17%; post = 6-14%).
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Effects of yohimbine on cutaneous reflex excitability
After yohimbine (800-1,600 µg/100 µl), only minor changes in the reflex response to electrical stimulation were documented in both intact (9/9 trials) and spinal (4/4 trials) cats. Figure 16 shows an example of the mean response with SE (thin lines) in flexor and extensor muscles to electrical stimulation of the contralateral superficial peroneal nerves, before and after yohimbine (1,600 µg/100 µl) in the intact cat NG5 (Fig. 16A) and in the late spinal (Fig. 16B). In the intact cat (Fig. 16A) after yohimbine, the same stimulating current used in control (230 µA) caused similar short-latency responses except in the hip flexor muscle (RSrt) albeit a decrease was found. Also the SE of the mean were usually smaller after yohimbine. In the spinal cat (29 days), the same stimulation used before yohimbine, produced a similar amplitude in the response for both flexors Srt and TA muscles and a decrease in the knee extensor VL muscle (Fig. 16B). These changes observed in some muscles after yohimbine were minor when compared with those observed following clonidine (Fig. 8). Figure 16C shows an example of a fast paw shake response before and after a high dose of yohimbine (1,600 µg/100 µl) in cat NG3, 18 days postspinalization. After 20 min following yohimbine administration, the fast paw shake was present but its duration was shorter.
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In summary, high doses of yohimbine (800-1,600 µg/100 µl) in intact cats caused major walking difficulties characterized by asymmetric stepping, stumbling, and a poor lateral stability. Small doses (400 µg/100 µl) induced slight effects such as abduction of one of the hindlimbs with an increase of contralateral EMG amplitude leading to the turning of the hindquarters to one side or the other. Also, following yohimbine injection, intact cats had major difficulties to walk on slopes and on the ladder; these effects lasted from 2 to 30 min. In the spinal state, yohimbine had no effect even at large doses. No major change in the cutaneous excitability was observed after yohimbine if compared with those observed after clonidine in both conditions.
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DISCUSSION |
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The purpose of this study was to compare the effects of the
2 agonist clonidine and the antagonist
yohimbine in the same cats, initially in the intact state and after
spinalization, to determine how the effects of these drugs vary
according to the animal's condition.
Clonidine injection on locomotion in intact and spinal cats
The differential effects of clonidine in intact versus spinal cats
can only be observed in the late spinal cat, when the cat was capable
of hindlimb locomotion on the treadmill. Whereas activation of
2-adrenoceptors in the intact cat decreased
step cycle duration and sometimes burst duration in the spinal cat, the
step cycle duration, in particular the swing phase, increased after
clonidine together with an augmented burst duration and amplitude. In
both intact and spinal cats, activation of
2-adrenoceptors by clonidine seemed to
modulate essentially the timing of the muscles and to a much lesser
extent the output amplitude. As proposed before, clonidine may exert an
effect primarily on interneurons that coordinate the timing between
flexor and extensor muscles (Chau et al. 1998a
).
In the intact cat, the small transient effects seen after clonidine or
NA on locomotion indicate that animals can readily compensate for the
imbalance of one system, namely in this case an over stimulation of
receptors. Furthermore, in the intact cat after clonidine, the
regularity of walking was improved, as reflected by a much more
homogeneous and better-organized EMG pattern. This finding was also
observed in the same animal after spinalization as well as in other
studies where clonidine improved the characteristic walking pattern
that had deteriorated and had become irregular due to a lack of
training (Rossignol et al. 1995). This improved regularity of walking documented after clonidine in both intact and
spinal conditions may be the manifestation of changes occurring in the
properties of rhythm generation circuits. It had been shown that L-DOPA
and clonidine induce plateau potentials in spinal motoneurons in acute
complete spinal cats (Conway et al. 1988
). It has been
proposed that the role of the plateau potentials was to facilitate,
shape, and time the propagation of motor rhythm (Kiehn et al.
1996
). Thus this could lead to the more regular and constant
locomotor pattern seen after clonidine. It is also possible that the
regularization of the rhythm by clonidine in the intact condition was
due to side effects such as drowsiness. In this state, the cat could be
less disturbed by external stimuli and therefore reduce the
irregularity. However, this could not explain the improvement of
regularity documented in the spinal condition after clonidine.
The effects of clonidine in the early and late spinal cat reported in
this study are in close agreement with previous results (Barbeau
et al. 1987; Chau et al. 1998a
). These authors
have previously shown that clonidine can initiate locomotion in the
early spinal cats and in a few week after the spinalization (i.e., late
spinal cats), modulate the locomotor pattern by increasing the cycle duration and flexor burst duration while the mean EMG amplitude tended
to increase or remain the same in flexor and decrease in extensors.
However, in the present experiment, some increase in extensor bursts
amplitude was sometimes seen after high doses of clonidine (Fig.
4B). This may result from activation of
1-adrenergic receptors at high doses of
clonidine (Kehne et al. 1985
; Timmermans and van
Zwieten 1982
) since their stimulation was found to increase the
output amplitude of extensor muscles to a much greater extent than
following the
2-noradrenergic receptor
stimulation (Chau et al. 1998a
). These results, in both
intact and spinal cat, are also in contrast to findings in cats
subjected to ventral and ventrolateral spinal lesions in which
clonidine had a detrimental effect on walking (Brustein and
Rossignol 1999
). In these partial spinal cats, clonidine caused
major reduction in weight support of the hindlimbs, an increase in
swaying of the hindquarters, stumbling, and falling.
The differential effects of clonidine in the intact cat compared with
complete and partial spinal cats could in part be attributed to a
difference in targets, alterations in the sensitivity of 2 receptors as well as to temporal
modifications in receptor densities that follow spinal lesions. In
intact cats, clonidine mediates its effect on both pre- and
postsynaptic
2-adrenergic receptors. After a
complete spinal cord transection, there is a degeneration of all
descending nerve fibers including NA-containing terminals, leading to a
loss of presynaptic
2-adrenergic receptors. In
such cats, clonidine mediated its effects mainly through activation of
the postsynaptic
2 receptors (Langer
1977
; Marshall 1983
). Differential effects on
flexor reflexes in both intact and spinalized rats have also been
reported after
2-adrenergic agonist tizanidine administration and could result to changes in targets in intact and
spinal states (Chen et al. 1987
). Furthermore, it has
been shown that
2 receptors densities differed
with time after the transection (Giroux et al. 1999b
).
At a short interval, i.e., 15-30 days, labeling increased below the
level of the transection, indicating that the denervation caused
changes in the affinities or led to receptor upregulations. These
changes could be related to some of the plastic modifications occurring
during that period, namely the recovery of locomotion. This
upregulation remained elevated until 30 days, but with time receptor
levels gradually returned to control values. Even if receptor densities
become normal in the late spinal cat, there may be modifications in the efficacy of the effector transducing mechanisms downstream of the
binding sites so that the receptor and effector complex may operate
more efficiently than under control condition. In the case of cats with
partial lesions, presynaptic
2-adrenoceptors may still be present on the spared NA descending terminals and could
thus contribute to the observed detrimental effect (Timmermans and van Zwieten 1982
).
Effects of yohimbine on locomotion in intact versus spinal cats
In this study, the specific 2 antagonist
yohimbine was used to selectively block
2-adrenoceptors (Goldberg and Robertson 1983
). Yohimbine has been shown previously to completely
antagonize the behavioral effect of clonidine on spinal locomotion
(Barbeau et al. 1987
). In the present study, yohimbine
did not affect the locomotion in spinal cats even if
2-adrenoceptors remain after spinal cord
transection (Giroux et al. 1999b
). Following a complete spinal cord transection in the cat, NA contents below the lesion decrease dramatically (Roudet et al. 1993
) and
antagonists mediate their effects only in presence of NA or NA
agonists; this may explain the lack of effects of yohimbine in the
spinal cat. Thus spontaneous spinal locomotion probably does not depend
on the activation of NA receptors since spinal cats can walk in the
absence of noradrenergic descending pathways, and blockade of
2-noradrenergic receptors with yohimbine does
not prevent spinal locomotion. The expression of spinal locomotion must
depend on other neurotransmitter systems located within the spinal
cord. In contrast, blockade of
2-noradrenergic
receptors in the intact cat had a detrimental effect on locomotion.
Thus the NA descending system probably contributes to the modulation of
this spontaneous locomotion.
The 2-adrenoceptors are autoreceptors on NA
nerve terminals that diminish neurotransmitter release; their blockade
by antagonist (e.g., yohimbine) will increase NA release
(Reimann and Schneider 1989
). Therefore this increased
NA may be responsible for the detrimental effect of yohimbine in the
intact cat. This interpretation is less likely since NA administration
in the intact cat (unpublished observations) had no such effect on
locomotor patterns. In Xenopus laevis, neuromodulators such
as NA and serotonin (5-HT) act presynaptically on glycinergic terminals
of commissural interneurons mediating reciprocal inhibition during
swimming (McDearmid et al. 1997
). If the same control
exists in higher vertebrates, yohimbine may interfere with the
presynaptic control of glycinergic release. This may cause alterations
of interlimb coordination, leading to the effect observed after
yohimbine administration in the intact cat, such as asymmetry of
stepping and turning of the hindquarter on one side.
Another explanation for the detrimental effects of yohimbine in intact
cats is that it may interfere with spinal interneurons that receive
influences from descending pathways controlling posture and balance.
Various brain stem and cerebellar regions have been so far implicated
in the control of posture. Lesions of the lateral and superior
vestibular nucleus, or of the associated fastigial nuclei of the
cerebellum, produced severe disturbances of posture and balance
(Carpenter et al. 1959; Modianos and Pfaff
1976
). Rats with such lesions had pronounced tremor and
abnormal head posture and exhibited an asymmetrical trunk posture; the
latter manifested by the tilting of the head and splaying of the limbs on one side of the body. Other descending pathways from the brain stem
reticular formation influence postural tonus and also have an effect on
posture and movement (Luccarini et al. 1990
; Mori 1987
; Takakusaki et al. 1994
). In spinal cats,
the transection destroys all descending fibers, including vestibulo-
and reticulospinal pathways; thus the experimenter has to provide
equilibrium and posture by gently steering the tail and maintaining the
cat straight on the treadmill.
Cutaneous excitability
In both intact and spinal cats, clonidine caused a decrease in the
fast paw shake responses as well as an increase in the threshold for
electrical stimulation. This decrease in cutaneous reflex excitability
was in line with previous studies in chronic spinal cats, showing that
clonidine reduced dramatically cutaneous reflex responses
(Barbeau et al. 1987; Chau et al. 1998a
).
Also, in fictive preparations,
2 agonists have
been reported to reduce fast paw shake response, i.e., high-frequency
synchronous activity of flexor and extensor muscle (Pearson and
Rossignol 1991
). Short latency transmission from afferents has
been found to be depressed after L-DOPA and clonidine
(Andén et al. 1966
; Grillner 1973
) as well as by stimulation of the mesencephalic locomotor region, known
to trigger locomotion in the decerebrate cat (Grillner and Shik
1973
). In awake, nonanesthetized monkeys, Corboz et al.
(1991)
showed that a
2 agonist reduced
the EMG response of the flexor reflex induced by stimulation of
cutaneous afferents, and this could be prevented by the administration
of yohimbine, suggesting that it is mediated by an
2-adrenoceptor. Other studies have reported
that NA agonists, including clonidine, depress transmission from group
II muscle afferents in cats (Bras et al. 1990
;
Schomburg and Steffens 1988
). Furthermore, stimulation
of the locus coeruleus and subcoeruleus in decerebrate cats depressed
interneuronal pathways involved in the reflex action of group II
afferents on motoneurons (Jankowska et al. 1993
), and NA
was found to modulate ascending information originating from skin and
muscles afferent (Jankowska et al. 1997
).
In intact and spinal cats, clonidine seems to affect cutaneous reflex
excitability in a similar way; the threshold for electrical stimulation
in both conditions increased three to four times after its
administration. However, the time course of its effect seems to be
shorter in the intact cat; i.e., cutaneous responses return to normal
after 2-3 h in the intact cat while in the spinal animal some of these
effects are still present 6 h later. This difference in time
course could be due to the effective inactivation mechanisms or better
clearance mechanisms in intact cats. Although yohimbine did not affect
dramatically the cutaneous reflex excitability in both intact and
spinal cats, it was found to reverse the clonidine's decreased
cutaneous reflex response (Barbeau et al. 1987).
Slopes and walking on ladder
After clonidine, all cats were able to walk either on 15° slopes
(uphill or downhill) or on a horizontal ladder. On the ladder, they
could correctly place their feet on the round rungs but their paws
often slipped off. The decrease in the cutaneous excitability after
clonidine may be responsible for such sliding. In contrast, following
yohimbine administration the intact cats could not place correctly
their hindfeet on the rungs and had major difficulty walking on slopes
despite normal cutaneous excitability, suggesting effects on spinal
neurons that receive descending commands. Indeed, it has been shown
that destruction of the motor cortex, or interruptions of the
corticospinal tract, produced no major change on level walking but lead
to the inability of cats to walk on a wire mesh or on horizontal bars
(Eidelberg and Yu 1981). Moreover, walking on the ladder
was impossible after inactivation of the motor cortex by tetrodotoxin
or after cortical lesions (Beloozerova and Sirota 1993a
). Thus it is conceivable that blocking
2-adrenoceptors at the spinal level may have
interfered with spinal neurons that receive inputs from descending
supraspinal tracts, such as the corticospinal that control the accuracy
of locomotor movements. On the other hand, the corticospinal tract
seems to be less important during walking on slopes since the
inactivation of the motor cortex did not impede the performance of cats
walking on an incline surface (Beloozerova and Sirota
1993b
). Also, discharges of cortical neurons and pyramidal
tract fibers during locomotion up a 10° incline were the same as
during locomotion at an horizontal level (Armstrong and Drew
1984
). Yohimbine may act on interneurons that receive other
descending projections, such as the reticulospinal or vestibulospinal pathways; the cells of origin of these tracts have been shown to be
modulated with EMG activity during locomotion on an inclined surface
(Matsuyama and Drew 1996
) or even during level walking (Drew et al. 1986
).
Intrathecal cannula
There are many advantages to use an intrathecal cannula for drug delivery such as allowing for the immediate action of drugs as well as reducing systemic side effects. In the present study, we also found that such delivery system can remain in place and be effective for a long period of time, i.e., 2 yr (see Fig. 2, cat NG2). This system can be quite reliable since the results were reproducible for a given drug in the same animal and on different days (Fig. 2).
The location of the cannula's tips does not account for the turning of
the hindquarters on one side, particularly as seen following yohimbine
injection, or for the intensity of drug effects. In fact, the cannula
of cat NG3 terminated on the left side of the spinal cord
while the cannula's tip was found on the right side in the cat
NG5, and both cats turned on the same side. Furthermore, cat
NG3 was found to have the most rostral cannula's location (L2 segment), a region removed from the
motoneuron pool responsible for locomotion; in this animal, clonidine
and yohimbine effects were comparable to those observed in the two
others cats. This was confirm with a study on localized applications of
drugs at specific lumbar segments in the spinalized cats
(Marcoux and Rossignol 2000). These authors found that
clonidine topically applied or micro-injected in the upper lumbar
segment L3-L4 was
sufficient to trigger locomotion, and this locomotor activity could be
blocked or prevented by yohimbine. Together, these findings suggest
that future pharmacological therapies should focus to these specific spinal segments.
Conclusions
The present study indicates that the stimulation of the
noradrenergic receptors are not essential for the control of locomotion in the spinal cat because the spinal cat can recover hindlimb locomotion in absence of NA descending pathways, and the blockade of NA
receptors does not perturb spinal locomotion. Therefore it is proposed
that others classes of receptors still present after the spinal
transection such as excitatory amino acids (EAA) may also be involved
in the activation of locomotion (Chau et al. 1994;
Douglas et al. 1993
). We are currently investigating the
glutamatergic system, in particular the
N-methyl-D-aspartate (NMDA) receptors, on
receptors binding and on the expression of locomotion in the intact and
chronic spinal cat. Preliminary evidence suggests that glutamatergic
receptors are not downregulated several months after spinalization when
compared with other receptors (Giroux et al. 1999b
).
In contrast, in the intact state, the NA descending system plays a
crucial role, since blockade of
2-adrenoceptors results in a marked disruption
of walking. One possible role of the NA system is that in the spinal
cord both descending 5-HT and NA act as modulators that enhance the
effects of excitatory inputs to motoneurons. It has been demonstrated
that iontophoretic applications of 5-HT or NA on lumbar motoneurons
does not cause action potentials but rather dramatically potentiates
the action of glutamate-evoked action potentials (White and
Newman 1980
). The modulatory role of monoamines was later
confirmed in rat spinal cord preparations, where 5-HT and NA modulate
the NMDA-induced oscillatory activity of spinal cord motoneurons and
interneurons (Cazalets et al. 1990
; MacLean
et al. 1998
). If this applies to humans, then a strategy of
combining drugs that could interact, i.e., NA or 5-HT agonists with
other drugs acting on glutamatergic receptors, may be beneficial. Therefore knowledge on the neuropharmacology of the spinal cord injury
could be of great importance to provide a pharmacological basis for a
rational choice of therapeutic agents in the management of patients
suffering from impaired motor function after chronic spinal cord lesions.
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ACKNOWLEDGMENTS |
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We gratefully acknowledge J. Provencher and F. Lebel for assistance during surgeries, experiments, analyses, and preparation of the illustrations. We also thank P. Drapeau and G. Messier for programming, C. Gagner for electronic support, C. Gauthier and D. Cyr for the illustrations, J. Faubert for help during surgery, and J. Lavoie for histological assistance.
This work was supported by the Canadian Institutes for Health Research and the Spinal Cord Research Foundation (SCRF). N. Giroux was sponsored by studentships from the Neuroscience Network of Centers of Excellence of Canada, from the Rick Hansen Man in Motion Foundation (SJ-06), and from the Groupe de Recherche sur le Système Nerveux Central (GRSNC) of the Fonds Pour la Formation de Chercheurs et l'Aide à la Recherche.
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FOOTNOTES |
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Address for reprint requests: S. Rossignol, Dept. de Physiologie, CRSN, Faculté de Médecine, Université de Montréal, C.P. 6128, Succursale Centre-ville, Montreal, Quebec H3C 3J7, Canada (E-mail: serge.rossignol{at}umontreal.ca).
Received 30 June 2000; accepted in final form 9 February 2001.
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REFERENCES |
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