Centre de recherche en Sciences Neurologiques, Faculté de Médecine, Université de Montréal, Montreal, Quebec H3C 3J7, Canada
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ABSTRACT |
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Brustein, Edna and
Serge Rossignol.
Recovery of locomotion after ventral and ventrolateral spinal
lesions in the cat. II. Effects of noradrenergic and serotoninergic drugs. The effects of serotoninergic and noradrenergic drugs
(applied intrathecally) on treadmill locomotion were evaluated in two
adult cats subjected to a ventral and ventrolateral spinal lesion
(T13). Despite the extensive spinal lesion, severely
damaging important descending pathways such as the reticulo- and
vestibulospinal tracts, both cats recovered quadrupedal voluntary
locomotion. As detailed in a previous paper, the locomotor recovery
occurred in three stages defined as early period, when the
animal could not walk with its hindlimbs, recovery period,
when progressive improvement occurred, and plateau period,
when a more stable locomotor performance was observed. At this latter
stage, the cats suffered from postural and locomotor deficits, such as
poor lateral stability, irregular stepping of the hindlimbs, and
inconsistent homolateral fore- and hindlimb coupling. The present study
aimed at evaluating the potential of serotoninergic and/or
noradrenergic drugs to improve the locomotor abilities in the early and
late stages. Both cats were implanted chronically with an intrathecal
cannula and electromyographic (EMG) electrodes, which allowed
determination, under similar recording conditions, of the locomotor
performance pre- and postlesion and comparisons of the effects of
different drugs. EMG and kinematic analyses showed that
norepinephrine (NE) injected in early and plateau periods
improved the regularity of the hindlimb stepping and stabilized the
interlimb coupling, permitting to maintain constant locomotion for
longer periods of time. Methoxamine, the
1-agonist (tested only at the plateau period), had
similar effects. In contrast, the
2-agonist,
clonidine, deteriorated walking. Serotoninergic drugs, such
as the neurotransmitter itself, serotonin (5HT), the
precursor 5-hydroxytryptophan (5HTP), and the agonist
quipazine improved the locomotion by increasing regularity
of the hindlimb stepping and by increasing the step cycle duration. In
contrast, the 5HT1A agonist 8-hydroxy-dipropylaminotetralin (DPAT) caused foot drag in one of the cats,
resulting in frequent stumbling. Injection of combination of
methoxamine and quipazine resulted in maintained, regular stepping with
smooth movements and good lateral stability. Our results show that the
effects of drugs can be integrated to the residual voluntary locomotion and improve some of its postural aspects. However, this work shows clearly that the effects of drugs (such as clonidine) may depend on
whether or not the spinal lesion is complete. In a clinical context,
this may suggest that different classes of drugs could be used in
patients with different types of spinal cord injuries. Possible
mechanisms underlying the effect of noradrenergic and serotoninergic
drugs on the locomotion after partial spinal lesions are discussed.
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INTRODUCTION |
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Adult cats subjected to a massive bilateral
ventral and ventrolateral spinal lesion at the lowest thoracic level,
severely damaging ascending and descending pathways, among others, the reticulospinal and vestibulospinal pathways, can recuperate voluntary quadrupedal locomotion (Bem et al. 1995; Brustein
and Rossignol 1998
; Gorska et al. 1993a
,b
;
Rossignol et al. 1996
). However, as described in detail
in a preceding paper (Brustein and Rossignol 1998
), the
cats suffered from transient as well as from long-lasting postural and
locomotor deficits, the severity of which depended on the extent of the
spinal lesion. Early after the lesion (early period), none
of the cats could walk or support the weight of their hindquarters, and
they moved around using only the forelimbs. During the recovery
period, which lasted from 3 days up to 3 wk depending on the
extent of the spinal lesion, the cats gradually regained the ability to
support weight and walk with their hindlimbs. However, even when having
reached a plateau period during which there was no further
improvement, the quadrupedal stepping overground and on the treadmill
remained wobbly with poor lateral stability. The coupling phase between
the homolateral fore- and hindlimb was highly variable, and, in the
most lesioned cats, the fore- and the hindlimbs could walk at different
frequencies, which caused continual deviations in the coupling phase
between the two girdles. These locomotor deficits often resulted in
stumbling or falling on one side, which limited the cats to only a few
consecutive steps at a time and to low treadmill speeds (0.1-0.35
m/s). These locomotor deficits were more pronounced during walking
uphill or downhill on inclined treadmill (10°), and the amplitude
modulation (AM) of the hindlimb electromyographic (EMG) activity,
usually seen when walking uphill, was absent.
The most extensively lesioned cats did not improve their locomotor skills very much with time and expressed these deficits even 300 days postlesion. It was suggested that after functional and anatomic reorganization, the remaining pathways in the dorsolateral funiculi, such as the corticospinal, the remaining reticulospinal, and possibly propriospinal, were sufficient to provide the necessary drive for initiation of locomotion as well as some postural control. However, their contribution is limited because the cats cannot maintain constant fore- and hindlimb coupling or adapt to more demanding situations such as inclined walking.
It is clinically relevant to try and improve the locomotor capacities
in this animal model of incomplete spinal lesion because the relative
percentage of partial versus complete spinal lesions is increasing in
the population of patients (Tator et al. 1993). Patients
with partial spinal lesions exhibit residual locomotor capacities but
suffer from locomotor and postural deficits such as difficulties in
bearing weight, maintaining balance, and adapting their walking speed
(Barbeau and Rossignol 1994
). Therefore the purpose of
this study was to try and improve the residual voluntary locomotor
function by applying different noradrenergic (NE) and serotoninergic
(5HT) drugs through a chronic intrathecal cannula. These two groups of
drugs were chosen because of their known involvement in initiation
and/or modulation of locomotion in complete spinal cats (Barbeau
and Rossignol 1994
; Barbeau et al. 1993
;
Chau et al. 1998a
,b
).
The specific purpose of this study was then to investigate the effects of noradrenergic and serotoninergic drugs on the treadmill locomotion of two cats subjected to extensive but partial spinal lesions of the ventral and ventrolateral quadrants. We wanted first to try and express locomotion in the early period after the lesion when there is practically no hindlimb locomotion and second to improve the locomotor capacities (walking maintenance and regularity, coupling between the fore- and the hindlimbs and speed adaptation) at a later period when voluntary quadrupedal walking was reestablished but is deficient.
Therefore, the different drugs were applied in two cats through an intrathecal cannula with its tip ending caudal to the spinal site of lesion. The locomotor activity was compared before and after the drug application using chronically implanted electrodes to identify changes in EMG activity under constant recording conditions in the same cat and to follow the progressive evolution of its locomotor capacities during the recovery period up to the point of achieving a stable locomotor behavior (plateau period).
Our results show that some drugs (noradrenaline, methoxamine, and quipazine) improved the walking capacities of the cats, resulting in a more stable and maintained locomotor performance. However, other drugs, such as clonidine, caused a deterioration of the locomotor performance. The mechanisms for the action of the drugs in partial spinal cats are discussed with reference to clinical situations.
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METHODS |
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General experimental protocol
Experiments were carried out on two adult cats (EB7
and EB8) weighing 4.1 and 2.8 kg, respectively, which were
among the most lesioned cats reported in our previous study
(Brustein and Rossignol 1998). The cats first were
trained to walk on a treadmill at different speeds (0.2-0.7 m/s) until
they could maintain a regular locomotion for periods of ~20 min. Then
the cats were anesthetized and implanted with EMG electrodes and an
intrathecal cannula. Control experiments (n = 7-12)
were made during a period of 1-6 wk to obtain EMG and kinematic values
in the intact state. Thereafter the cats were submitted to the ventral
and ventrolateral spinal lesion at the thoracic level
(T13), and their locomotor recovery was followed and
documented daily as well as long after no further recovery could be observed.
Different adrenergic and serotoninergic drugs were tested, and their effects on locomotion were followed for several hours and compared with the performance of the cat immediately before drug application.
At the end of the experimental series, wheat germ agglutinin-horseradish peroxidase (WGA-HRP) was injected caudal (L2) to the site of spinal lesion (at days 340 and 280 postlesion for cats EB7 and EB8, respectively). Then 3 days later, the cats were perfused and the spinal site of lesion and the spinal site of WGA-HRP injection as well as the brain stem and the motor cortices were processed histologically. All the surgical procedures and experimental protocols were reviewed and approved by the University's Ethics Committee.
Surgical procedures
During the first surgery, performed in sterile conditions and under general anesthesia, a cannula was inserted into the intrathecal space of the spinal cord and EMG electrodes were implanted in various fore- and hindlimbs muscles.
EMG IMPLANTATION.
The surgical procedure for implantation of EMG electrodes and the
function of the implanted muscles were detailed in the preceding paper
(Brustein and Rossignol 1998). The implanted muscles
included, in the hindlimbs: iliopsoas (Ip) and sartorius (Srt),
semitendinosus (St), tibialis anterior (TA), vastus lateralis (VL),
and gastrocnemius medialis (GM) and lateralis (GL); in the forelimbs:
cleidobrachialis (ClB) and the lateral head of the triceps brachii
(TriL). Although all muscles were implanted both on the left (L) and on
the right (R) limbs, only the left side of the animal facing the video
camera was used to illustrate the kinematics.
INTRATHECAL CANNULA. A Teflon cannula (Teflon tube-thinwall, size 24 gauge) was inserted through an opening made in the atlanto-occipital ligament and gradually pushed through the intrathecal space, so that the tip was located at L4-5, as measured before the insertion using external landmarks. The cephalic end of the cannula was inserted into a right-angle plastic port fixed to the skull by dental acrylic, next to the EMG connectors, and served for administration of drugs or sterile saline (0.9%). The inlet was capped to reduce contamination and was opened only at the time of drug or saline application. The dead space of the cannula was measured before the insertion and was determined to be ~100 µl. To ensure its patency, the cannula was flushed with a bolus of 100 µl sterile saline solution on nonexperimental days.
VENTRAL AND VENTROLATERAL SPINAL LESION.
The ventral and ventrolateral spinal lesions were performed in a second
surgical intervention, under general anesthesia, when the recordings of
the control period were completed (see Brustein and Rossignol
1998).
Recordings and data analysis
EMG activity and the video images used for kinematic analyses
were recorded simultaneously. They were synchronized using a SMPTE time
code (time code generator Skotel TCG-80N and time code reader TCR-80N),
recorded on both analog and video tapes (for details, see
Brustein and Rossignol 1998).
In addition to quantitative kinematic analysis, the video tapes were reviewed frequently to obtain a better overall impression of the drug effects because the kinematic analyses in one plane gives only a partial assessment of the locomotor improvement. In some cases, the cats also were filmed walking from above to better evaluate the body orientation during walking and its improvement after drug injections (not illustrated).
Drug application
The tested drugs are listed in Table 1 together with their main action and the given doses. All the drugs were administered to both cats and were generally repeated in at least three separate experiments at different days after the partial spinal lesion (also indicated in the rightmost columns in Table 1).
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Before drug application, a walking sequence on the treadmill was
recorded to determine the predrug baseline performance, which varied
with time after the lesion (see Brustein and Rossignol 1998). Then a bolus of 100 µl of the drug solution, prepared
with sterile saline (0.9%), was administered through the intrathecal cannula using an adapted syringe to fit the inlet. The syringe was
fastened to the rostral end of the cannula to avoid leakage and to
ensure that the whole dose would be injected. Immediately afterward, a
second bolus of 100 µl sterile saline was injected and served to
flush the drug out of the dead space of the cannula. Usually one dose
of one drug was given, unless indicated otherwise, and consecutive drug
experiments were separated by
48 h. The doses were kept as small as
possible to minimize central effects or any other discomfort to the
cat. The reaction time to the application of a drug, its maximal
effect, and the fading time depended on the drugs. To cover the whole
time course of the effects, recordings started as soon as 20-30 min
after the drug application and were repeated each 30-45 min until the
drug effects wore off (between 2 and 5 h). The results presented
illustrate the maximal effects of the drug on any given day. The drugs
were applied during the early, recovery, and plateau periods postlesion
(see Table 1); however, the illustrated experiments were recorded in
the early period when the cats had pronounced locomotor deficits and
during the plateau period when the cats exhibited consistent locomotor deficits. It should be emphasized that the duration of the different periods and the severity of the deficits depended on the extent of the
spinal lesion (see Brustein and Rossignol 1998
). For
example, for the most severely lesioned cat (EB7), the early
period lasted almost 4 wk, whereas it lasted only 1 wk for cat
EB8.
Evaluation of the extent of the spinal lesion
Evaluation of the extent of the spinal lesion was done by
combining conventional histological staining methods (cresyl violet, Kluver-Barrera) and by retrograde WGA-HRP labeling. The histological procedures were detailed in Jiang and Drew (1996),
Matsuyama and Drew (1997)
, and also in the companion
paper (Brustein and Rossignol 1998
).
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RESULTS |
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Extent of the spinal lesion and recovery of locomotion
The results of the histological evaluation of the spinal lesions
of cats EB7 and EB8 were described in detail in
the companion paper (Brustein and Rossignol 1998) (a
summary is provided in Fig. 1). Briefly,
examination of the site of spinal lesion of cat EB7 showed a
major disruption of both ventral and ventrolateral funiculi. Intact
tissue could be found only in the dorsal columns and in the left
dorsolateral funiculus (DLF) (see Fig. 1A for the
photomicrograph of the site of spinal lesion). The damage to the spinal
quadrants was correlated with a pronounced decrease in the numbers of
HRP-labeled neurons (expressed as percentage of the intact, Fig.
1A) found in brain stem nuclei corresponding to the origin
of reticulo- and the vestibulospinal pathways descending in these
quadrants (see Table 1 in Brustein and Rossignol
1998
, for the number of HRP-labeled cells). The lesion of
cat EB8 (Fig. 1B) encompassed the lateral
vestibulospinal pathway, bilaterally, whereas the reticulospinal
pathways were damaged but to a lesser extent compared with cat
EB7. In both cats, a major increase was observed in the number of
HRP-labeled cells in the motor cortex contralateral to the less
affected DLF (Fig. 1, A and B), as well as by
changes in the distribution of the cells (Brustein et al. 1997
).
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Despite the extensive lesion to the ventral and ventrolateral spinal
quadrants damaging important reticulo- and vestibulospinal pathways,
both cats recovered voluntary quadrupedal locomotion. Yet they suffered
from long-lasting locomotor deficits, as illustrated in Fig.
2. From the consecutive stick figure
diagrams and the angular excursion traces, it is noted that even at
~120 days postlesion (see Fig. 2, B and D), the
hindlimb stepping was highly irregular compared with the intact
situation (Fig. 2, A and C). The cats also
suffered from poor lateral stability and sudden stumbling, which are
reflected in abrupt amplitude increases of the EMGs bursts (see LSrt of
cat EB7 and RSrt for cat EB8). In addition, the
homolateral fore- and hindlimb coupling was affected as illustrated for
the most lesioned cat, EB7, in Fig. 4A and in the
phase plots of Fig. 8B. The coupling phase between the
fore-and hindlimb shifted gradually, a result of different walking
rhythms in the fore-and the hindlimbs. For example, in Fig.
2B, six bursts were counted in RTriL or in LTriL compared
with only five bursts in LVL. In cat EB8 the fore- and the
hindlimbs stepped at the same frequency and the coupling shifts were
within the range of one-step cycle duration. The ability to maintain a
constant homolateral fore- and hindlimb coupling was permanently
deficient for cat EB7 and transient for cat EB8. Cat
EB8 not only maintained a more constant interlimb coupling but
generally did better long-term postlesion compared with cat
EB7. For example, cat EB8 could follow higher treadmill
speeds of 0.5 m/s, whereas cat EB7 could hardly walk at
0.4 m/s, and its daily performance was much more variable.
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Drug applications
NE INJECTION IN THE EARLY PERIOD POSTLESION. The effects of NE application, early after the spinal lesion, are illustrated in Fig. 3 (cat EB7), and the mean changes in the EMG activity (duration and amplitude) are summarized for both cats in Table 2.
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Application of NE during the plateau period
The injection of NE in cat EB7 was repeated further during the plateau period (this experiment was not done in cat EB8). A representative example for such an experiment is given in Fig. 4 and summarized in Table 3.
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Before the drug (Fig. 4A), the cat could follow a treadmill speed of 0.3 m/s. But its walking pattern (compared with the intact state, see Figs. 3A and 2A) was irregular as reflected in the abrupt changes in the joint angular traces and in the variability of the EMG activity. The coupling phase between the fore- and the hindlimbs was also variable as illustrated by the dashed lines connecting the foot falls of the homolateral limbs. As with application of NE in the early period (Fig. 3), NE improved locomotion also when given during the plateau period. In the two situations, the stepping of the hindlimbs became much more regular (see foot fall diagrams and smoother angular excursion traces in Fig. 4B) and there was a general increase in the EMG amplitude (see Table 3). However, there were some differences between NE effects in the early and the plateau periods. For example, during the plateau period, the increase in the joint angular excursion was limited to the knee joint only (from 12 to 34°) and more importantly, NE stabilized both the coupling pattern and the phase drifts between the homolateral fore- and the hindlimbs (see foot falls diagrams in Fig. 4B) to a pattern which resembles the intact one (Fig. 3A).
Clonidine
In light of the improvement in the walking after injection of NE,
we further tested the 2-noradrenergic agonist, clonidine because it is implicated in the initiation and the modulation of
locomotion in the completely transected spinal cats (for review see
Rossignol 1996
). The effects of intrathecal injection of
clonidine in the early period were quite different from those observed
in the complete spinal cat, and they were also different from the effects of NE in the partial spinal cat at that period (see Fig. 3).
Not only clonidine did not improve the locomotor capability of the
cats, but it appeared to impair it. However, because the predrug
pattern was highly deficient (see Fig. 3B) it was difficult to assess the changes quantitatively.
In the plateau period, the adverse effects of clonidine were very obvious in both cats. At that stage, even one bolus of clonidine (0.8-1.9 mM/100 µl) was sufficient to cause a major reduction in the weight support of the hindlimbs, to increase swaying of the hindquarters, stumbling, and falling, which limited the cats to a few steps at a time at very low treadmill speeds (0.2 m/s). The effects of clonidine on the locomotion of cat EB7 are illustrated in Fig. 5B.
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Forty-five minutes after injection of clonidine (1.9 mM/100 µl) the cat could barely support its hindquarters and could hardly step. As illustrated in Fig. 5B, the stick figure diagram, the joint angular excursion traces and the EMG activity, are extremely disorganized (see Fig. 6A for the average changes in EMGs amplitude). These detrimental effects were long-lasting and the return to predrug walking capacities could not be observed for the following five recording hours.
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The effect of clonidine could be reversed mostly by an injection of
yohimbine (2-noradrenergic antagonist). As illustrated in Fig. 5C, 60 min postinjection of yohimbine (20.5 mM/100
µl), given 45 min postinjection of clonidine, thus well within the period of maximal action of clonidine (Fig. 5B), the weight
support of the hindquarters improved and so did the regularity of the stepping, and the cat's ability to walk at higher speeds (
0.3 m/s).
Yohimbine also improved the regularity of the EMG pattern, which
returned to predrug level (for quantitative effects on the EMG activity
in both cats, see Fig. 6).
Methoxamine
Because there was a major locomotor improvement after injection of
NE, but none with the 2-noradrenergic agonist,
clonidine, we further tested the effects of an
1-noradrenergic agonist, methoxamine on the locomotion
(applied only during the plateau period). The results of such
experiments, are illustrated in Figs. 7
and 8, and the EMG values are
summarized in Fig. 9. These results with
methoxamine were reproduced in both cats in all the experiments (for
details, see Table 1).
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Methoxamine considerably improved the locomotion of cat EB7. An hour and 30 min after application of 4 mM/100 µl methoxamine, the steps were more robust and regular, with forceful foot placement and were performed with a better lateral stability. The regularity of the steps is noted clearly when comparing the consecutive stick figures diagram before (Fig. 7A) and after methoxamine (Fig. 7B). The related angular excursion traces show that the movements of the joints are smoother and reproducible from cycle to cycle because all the abrupt changes characteristic of the predrug walking disappeared. The average range of movement at the hip, ankle, and MTP joints decreased after the application of methoxamine (from 20 down to 14°, 33 to 22°, and from 57 to 44°, respectively) whereas a slighter decrease was observed in the range of the knee movement (from 27 to 23°). The activity of the hindlimb muscles markedly changed. There was a general increase in the normalized amplitude compared with the predrug situation (see especially RGM). In addition, the duration of LSt increased significantly (see average EMG values in histograms of Fig. 9).
After the administration of methoxamine, the ability of the cat to follow higher treadmill speeds did not improve. Nevertheless there was a major improvement in the ability of the cat to maintain locomotion at the same speed range for longer periods of time. Predrug, the cat could make about 6 consecutive steps, whereas after application of methoxamine, it easily did 20 steps.
The coupling phase between the homolateral fore-and hindlimb also changed after the drug application, as illustrated in Fig. 8. The graphs of the phase of burst onset of LTriL and LVL show that, predrug (Fig. 8B), the homolateral coupling phase varied between 0.4-0.8, whereas post methoxamine (Fig. 8C), it was within the intact range (Fig. 8A) and fluctuated around a value of 0.25. Further, the coupling pattern was changed from a diagonal type predrug (Fig. 8B) to almost an intact pattern with the difference that the forelimbs were leading. The general improvement in the locomotion after methoxamine was long-lasting and could be observed for 4-5 h.
Methoxamine improved the walking of cat EB8 as well (for the average EMG values see Fig. 9). As with cat EB7, the regularity of the hindlimbs stepping improved. This is indicated by a decrease in the variability of the hindlimb step cycle duration (variance ratio test P < 0.01, see mean values in Fig. 9D). There was also a stabilization of the coupling between the homolateral fore- and hindlimbs, around a phase value of 0.2, which resembles the intact situation. Methoxamine also caused a decrease in the average range of angular displacement in the hip, knee, and ankle joints (from 39 down to 33°, 38 to 26°, 30 to 19°, respectively), much like what was observed in cat EB7. These changes, in contrast to the observed in cat EB7, were accompanied by a decrease in EMG amplitude (see Fig. 9).
Serotoninergic drugs
Several serotoninergic drugs were tested: the neurotransmitter
itself, 5HT; the 5HT1,2,3 agonist, quipazine; the 5HT
precursor, 5-hydroxytryptophan (5HTP); and a 5HT1A agonist,
8-hydroxy-dipropylaminotetralin (DPAT). All these drugs, except the
agonist DPAT, improved locomotion in a similar manner. They differed
mainly in their time of action; 5HT and 5HTP did not last long (<2 h),
whereas the effects of quipazine could be observed for 5 h after the
application. DPAT was detrimental to locomotion of EB7 by inducing, as
soon as 20 min postapplication, a severe foot drag of both hindlimbs
causing the cat to stumble very often. This effect faded away after
1 h.
A representative example for the effects of quipazine on the locomotion of cat EB7 is given in Figs. 10 and 11, and the quantitative values of the EMG activity are summarized in the histograms of Fig. 12. Predrug, the cat walked irregularly, as illustrated in the stick figure diagram (Fig. 10A) showing five consecutive steps, each of a different duration. In addition, the angular movement of the joints was variable and included abrupt, sudden changes. The raw EMGs also reflected this disorganized walking. The EMG activity of the different muscles not only varied in amplitude and duration but also in their time course (see especially right and left GM).
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After injection of quipazine (1.2 mM/100 µl), the cat's walking improved considerably. It had better lateral stability and it could take long regular steps at a constant speed of 0.3 m/s (see Fig. 12), which were, still, shorter than the mean intact step cycle duration at 0.3 m/s (1,372 ± 62 ms). After the drug, the angular excursion traces were more regular and stable. In addition, there was a major increase in the average range of angular excursion of the hip, knee, and ankle: from 17 to 27°, 21 to 29°, and from 19 to 28°, respectively. The values of angular excursion, after quipazine, resembled more the intact values (26° in the hip, 32° in the knee, and 36° in the ankle). No change was observed in the average range of angular excursion of the MTP joint, which stayed lower relative to the intact. The EMG activity also reflected the increase in regularity and prolongation of the steps. There was a general tendency for prolongation of the burst duration relative to the predrug situation (see Fig. 12B), especially in the extensors. The latter was even significantly longer then the intact burst duration. Quipazine also stabilized the interlimb coupling, as illustrated in the average foot fall diagrams in Fig. 11. Predrug the coupling pattern was variable, as expressed by a continual shift between three main types of coupling patterns, homolateral in-phase coupling, diagonal coupling (Fig. 11A), and almost a normal coupling pattern (see average foot fall diagram of the intact in Fig. 8A). After quipazine, the cat was walking consistently with an homolateral, in-phase, coupling pattern, which is illustrated in the foot fall diagram in Fig. 11B, averaging 13 consecutive steps. This more robust coupling pattern probably resulted from an improved hindlimb stepping regularity (variance ratio test P < 0.01) and not from a better coupling between the fore- and the hindlimbs because even postdrug, the drift in the coupling phase persisted (see the related phase plot).
Combination of methoxamine and quipazine
As detailed in the preceding sections, both methoxamine and quipazine improved locomotion but in a different way. The first drug increased the amplitude of the EMGs, decreased the cycle duration and the joint angular excursion while the later prolonged the step cycle duration. Therefore both drugs were given at the same time in one bolus. The effects on stability of the walking is illustrated in Fig. 13, whereas the quantitative values are given in Fig. 14.
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The cat walking (regularity, maintenance, and adaptation to speeds) is illustrated in Fig. 13 by plotting the step cycle duration of a whole consecutive stepping sequence performed during one recording session. Before the drug (Fig. 13B), the cat had major difficulty maintaining a treadmill speed of 0.35 m/s for >10 step cycles, and after several steps in which it touched the back of the treadmill, the speed of the treadmill was decreased to 0.3 m/s. Even then the cat walked irregularly as reflected by a step to step fluctuation in the cycle duration.
Sixty-five minutes postapplication of one bolus of the combined
solution, which contained only half the doses (2 mM methoxamine + 1.2 mM quipazine/100 µl) used when each one of the drugs was tested
separately, an elegant and sustained walking was observed, with much
improved lateral stability. This is indicated by a much reduced
variability of the step cycle duration, which resembled generally the
intact situation (Fig. 13A). Notice, however, that in the
intact situation, walking at 0.3 m/s was somewhat too slow for this
cat, and this was expressed in higher step cycle variability. In
addition to improve regularity there was also an improvement in the
speed range the cat could follow and maintain (0.4 m/s). Generally,
the stepping was characterized by somewhat shorter steps (see Fig.
14B) and by a corresponding slight reduction in the average
range of the angular displacement (not illustrated). The EMG duration
and amplitude showed various changes as summarized in Fig. 14,
A and B.
The application of the combined solution also improved cat EB8's walking. In this cat we have observed an increase in the step cycle duration (see Fig. 14D). Changes also were found in the average range of angular excursion of the hip, ankle, and MTP, which decreased from 29 to 23°, 29 to 21°, and 48 to 43°, respectively. Significant increases were found in the amplitudes of all the flexors (see Fig. 14, C and D). The extensor amplitudes did not change, but their duration increased significantly compared with the predrug situation.
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DISCUSSION |
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In this work, we have demonstrated that different noradrenergic and serotoninergic drugs can improve or deteriorate walking of cats with partial but large ventral and ventrolateral lesions of the low thoracic spinal cord. First the extent of the spinal lesion with special emphasis on the damage to descending noradrenergic and serotoninergic tracts will be discussed. Then different possible mechanisms by which the drugs may exert their effects will be suggested as well as possible clinical applications.
Extent of the spinal lesions in relation to descending noradrenergic and serotoninergic pathways
The extent of the damage to noradrenergic and serotoninergic
pathways after the ventral and ventrolateral spinal lesions only could
be evaluated indirectly by comparing the results of the evaluation of
the extent of the spinal lesion (for details, see the preceding paper,
Brustein and Rossignol 1998) to what is known from the
literature about the noradrenergic and serotoninergic pathways in the
spinal cord.
Previous studies using histofluorescence and retrograde HRP labeling
showed that noradrenergic pathways originating in nucleus locus
coeruleus, the main source of spinal noradrenergic fibers, project
primarily through the ventrolateral funiculus. In addition, nucleus
subcoeruleus, nucleus Kolliker-Fuse, and cell bodies in A5 send fibers
through both the ventrolateral and the dorsolateral funiculi. The
noradrenergic fibers innervates the lumbar spinal cord bilaterally,
although the ipsilateral contribution dominates (Kuypers
1981; Kuypers and Maisky 1977
; Marshall
1983
; Stevens et al. 1985
). The serotoninergic
axons originating in the raphe nuclei, such as raphe pallidus and
obscuras, descend mainly in the ventral and ventrolateral funiculi,
whereas axons from raphe magnus descends primarily through the DLF
(Kuypers 1981
; Martin et al. 1978
).
According to these findings, most of the noradrenergic and the
serotoninergic axons descending in the ventral and ventrolateral quadrants of cat EB7 probably were damaged. However, the
noradrenergic and the serotoninergic axons descending in the left DLF
mostly were preserved. In cat EB8, the existence of
serotoninergic axons in left ventral quadrant cannot be excluded.
Nevertheless, an extensive damage probably was done to serotoninergic
and noradrenergic fibers descending in the lateral funiculi. The
difference in the extent of the spinal lesions (less severe for
cat EB8), which correlated with faster recovery and better
locomotor performance (see Brustein and Rossignol 1998),
may explain why generally larger doses of the tested drugs were needed
to see effects in cat EB8 long-term postlesion (see Table
1). Yet, and most importantly, the applied drugs showed overall similar
effects in both cats. Drugs such as clonidine had a detrimental effect
in both cats, and drugs that caused a pronounced improvement in the
walking of cat EB7 may have caused more subtle effects on
the walking of cat EB8, but they were never detrimental.
Noradrenergic drugs injection in the partial spinal cat
SUMMARY OF THE EFFECTS.
Injection of the neurotransmitter itself, NE, in the early period,
highly improved speed performance and stepping regularity of both cats.
It also improved the hindlimb joint angular excursion and the coupling
between the homolateral fore- and hindlimb. These effects were more
pronounced in cat EB7 than in cat EB8 most likely due to differences in their locomotor capacities, which related to the
extent of the spinal lesions. In the early period, NE improved the
stepping pattern considerably but not to the point that cats could walk
on their own with full-weight support and lateral stability of the
hindquarters. In the plateau period, however, the improvement in the
walking regularity was accompanied by a pronounced increase in the
weight support and lateral stability. The latter effects resembled the
effects of 1-agonist, methoxamine, and may be attributed to an increase in the extensor amplitude. Methoxamine, in contrast to
NE, caused a general decrease in the joint angular excursion, which may
explain why no improvement was observed in speed performance. Nevertheless, better stability, more regular hindlimb stepping, and
interlimb coupling may account for more sustained walking at the same
range of speeds. It is interesting to note that, after methoxamine,
cat EB8 showed more regular walking and a decrease in joint
angular excursion, as did cat EB7, but without major effects
on the amplitude of the recorded muscles.
PROPOSED MECHANISMS FOR THE ACTIONS OF THE DRUGS.
The improvement in locomotion of the partial spinal cat after NE and
methoxamine injections can be attributed to global changes in spinal
neurons excitability (Grillner 1981). According to this suggestion, the slight hyperpolarization caused by NE, a result of a
decrease in membrane conductance, will cause a general potentiation of
25-50% of other non-NE synaptic input. The possible implication of
noradrenergic drugs in mechanisms of gain amplification was suggested
as well by Hounsgaard et al. (1988)
and by Conway
et al. (1988)
. L-3-4-Dihydroxyphenyl-alanine
(L-DOPA) and clonidine induce plateau potentials in
spinal motorneurons of acute complete spinal cats (Conway et al.
1988
; Schomburg and Steffens 1996
), so does
methoxamine in the decerebrate cat preparation (Lee and Heckman
1996
, 1997
). Plateau potentials were proposed as a mechanism that reduces the need for sustained synaptic drive from the rhythm generator during locomotion. Therefore by maintaining excitability at a
constant level, only short-lasting excitation or terminating inhibition
is needed to start or stop the activity in the motorneurons (Conway et al. 1988
; Hounsgaard et al.
1988
). This may account for the major improvement in the
walking regularity after methoxamine and NE in both cats even without
major changes in EMG amplitudes as observed in cat EB8.
Hence NE and methoxamine probably provided a constant background
excitability on which the signals from the rhythm generator could be
expressed reliably and consistently.
Serotoninergic drugs injection in the partial spinal cat
SUMMARY OF THE EFFECTS. 5HT, the neurotransmitter itself, its precursor, 5HTP, and quipazine, a 5HT1,2,3 agonist, improved the lateral stability and the weight support of the hindlimbs, which resulted in a more regular quadrupedal walking with better interlimb coupling pattern, especially in the most lesioned cat, EB7. In addition, the serotoninergic drugs prolong the step cycle duration in both cats. In cat EB8, this prolongation was accompanied by an increase in flexors and by a slight increase in extensor burst duration, whereas in cat EB7, a significant increase in extensor burst duration was observed with a slight decrease in the normalized EMG amplitude. These effects were different from those observed after application of NE or methoxamine, which decreased the step cycle duration, joint angular excursion, and increased the amplitude of the EMGs.
The 5HT1A agonist, DPAT, had a detrimental effect on the locomotion of cat EB7, which was manifested as a severe foot drag in both hindlimbs, causing the cat to stumble often. However, compared with clonidine, this effect was short-lasting and was not accompanied by an apparent decrease of the weight support of the hindlimbs or an increased wobbliness.PROPOSED MECHANISMS FOR THE ACTIONS OF THE DRUGS.
In contrast to NE and clonidine, the 5HT precursor, 5HTP, was not found
to evoke locomotor rhythm in either the low spinal and decerebrate cat
(Grillner and Shik 1973) or in the complete chronic
spinal cat during the first week after spinalization, whereas clonidine
or L-DOPA caused a dramatic change in kinematics and EMG
pattern (Barbeau and Rossignol 1990
, 1991
;
Barbeau et al. 1993
). However, 5HTP markedly increased
the tonic activity in all muscles in both preparations (Barbeau
and Rossignol 1991
; Grillner and Shik 1973
). An
increase in extensor and flexor muscles activity (amplitude and
duration) also was observed in the chronic late spinal cat after
intraperitoneal injection of different serotoninergic drugs
(Barbeau and Rossignol 1990
, 1991
).
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ACKNOWLEDGMENTS |
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We gratefully acknowledge the contribution of J. Provencher and F. Lebel for assistance during surgeries, experiments, analyses, and preparation of the illustrations; P. Drapeau and G. Messier for programming; J. Lavoie, F. Cantin, and N. De Sylva for histological assistance; C. Gauthier and D. Cyr for illustrations and photographs; and C. Gagner for electronic support.
This work was supported by the Canadian Neuroscience Network and a group grant from the Medical Research Council of Canada. E. Brustein was supported by fellowships from the Canadian Neuroscience Network and the Groupe de Recherche sur le Système nerveux central (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, Centre de recherche en Sciences Neurologiques, Université de Montréal, P.O. Box 6128, Station Centre-Ville, Montreal, Quebec H3C 3J7, Canada.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 4 February 1998; accepted in final form 25 November 1998.
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REFERENCES |
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