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J Neurophysiol 79: 2941-2963, 1998;
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The Journal of Neurophysiology Vol. 79 No. 6 June 1998, pp. 2941-2963
Copyright ©1998 by the American Physiological Society

Effects of Intrathecal alpha 1- and alpha 2-Noradrenergic Agonists and Norepinephrine on Locomotion in Chronic Spinal Cats

Connie Chau1, Hugues Barbeau1, 2, and Serge Rossignol1

1 Centre de Recherche en Sciences Neurologiques, Faculté de Médecine, Université de Montréal; and 2 School of Physical and Occupational Therapy, McGill University, Montreal, Quebec H3G 1A5, Canada

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Chau, Connie, Hugues Barbeau, and Serge Rossignol. Effects of intrathecal alpha 1- and alpha 2-noradrenergic agonists and norepinephrine on locomotion in chronic spinal cats. J. Neurophysiol. 79: 2941-2963, 1998. Noradrenergic drugs, acting on alpha  adrenoceptors, have been found to play an important role in the initiation and modulation of locomotor pattern in adult cats after spinal cord transection. There are at least two subtypes of alpha  adrenoceptors, alpha 1 and alpha 2 adrenoceptors. The aim of this study was to investigate the effects of selective alpha 1 and alpha 2 agonists in the initiation and modulation of locomotion in adult chronic cats in the early and late stages after complete transection at T13. Five cats, chronically implanted with an intrathecal cannula and electromyographic (EMG) electrodes were used in this study. Noradrenergic drugs including alpha 2 agonists (clonidine, tizanidine, and oxymetazoline) and an antagonist, yohimbine, one alpha 1 agonist (methoxamine), and a blocker, prazosin, as well as norepinephrine were injected intrathecally. EMG activity synchronized to video images of the hindlimbs were recorded before and after each drug injection. The results show differential effects of alpha 1 and alpha 2 agonists in the initiation of locomotion in early spinal cats (i.e., in the first week or so when there is no spontaneous locomotion) and in the modulation of locomotion and cutaneous reflexes in the late-spinal cats (i.e., when cats have recovered spontaneous locomotion). In early spinal cats, all three alpha 2 agonists were found to initiate locomotion, although their action had a different time course. The alpha 1 agonist methoxamine induced bouts of nice locomotor activity in three spinal cats some hours after injection but only induced sustained locomotion in one cat in which the effects were blocked by the alpha 1 antagonist prazosin. In late spinal cats, although alpha 2 agonists markedly increased the cycle duration and flexor muscle burst duration and decreased the weight support or extensor activity (effects blocked by an alpha 2 antagonist, yohimbine), alpha 1 agonist increased the weight support and primarily the extensor activity of the hindlimbs without markedly changing the timing of the step cycle. Although alpha 2 agonists, especially clonidine, markedly reduced the cutaneous excitability and augmented the foot drag, the alpha 1 agonist was found to increase the cutaneous reflex excitability. This is in line with previously reported differential effects of activation of the two receptors on motoneuron excitability and reflex transmission. Noradrenaline, the neurotransmitter itself, increased the cycle duration and at the same time retained the cutaneous excitability, thus exerting both alpha 1 and alpha 2 effects. This work therefore suggests that different subclasses of noradrenergic drugs could be used to more specifically target aspects of locomotor deficits in patients after spinal injury or diseases.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Different neurotransmitters such as norepinephrine, serotonin, excitatory amino acids (EAA), and acetylcholine, have been identified to play a role in the initiation and modulation of locomotion in different animal preparations (for review, see Rossignol 1996). For example, in in vitro neonatal rat preparation, locomotor activity have been found to be released by EAA (Cazalets et al. 1990; Kudo and Yamada 1987; Smith and Feldman 1987), serotonin (Cazalets et al. 1992; Cowley and Schmidt 1994; Kiehn and Kjaerulff 1996), and cholinergic drugs (Katakura and Chandler 1991). In chronic spinal cats, among the different pharmacological agents, noradrenergic drugs were found to be the most effective in initiating locomotion (Barbeau and Rossignol 1991; Barbeau et al. 1987). The importance of the noradrenergic system has been shown by early studies from Lundberg and colleagues (Anden et al. 1966a,b; Jankowska et al. 1967) who demonstrated the ability of noradrenergic agents to activate neuronal circuits that could be responsible for locomotor function. They showed that intravenous injection of the noradrenergic precursor, dihydroxy phenylalanine (DOPA) inhibited the transmission of short latency responses from the flexor reflex afferent (FRA) but released long-latency and long-duration discharges not normally found in acute spinal cats. These late discharges often evolved as sequences of rhythmically alternating activity between flexors and extensors reminiscent of stepping. It was suggested indeed that the interneuronal circuitry generating the late discharges evoked after DOPA could be responsible for generating locomotion. This was pursued by Grillner and Zangger (1979) who showed that a detailed locomotor rhythm can be generated by the neuronal circuitry within the spinal cord itself. Indeed, after the injection of the noradrenergic precursor (DOPA) and nialamide (a monoamine oxidase inhibitor), a pattern of rhythmic alternating discharges in antagonist hindlimb muscle nerves was observed in acute spinal and curarized cat. DOPA (intravenously) has been postulated to mediate its effects through the activation of noradrenergic receptors (Anden et al. 1966a,b). Using a noradrenergic receptor agonist (clonidine), Forssberg and Grillner (1973) demonstrated the ability of noradrenergic drugs to initiate locomotion. They showed in acute spinal cats (Th12) that after an intravenous injection of clonidine, cats could walk with both hindlimbs when placed on a moving treadmill belt. They suggested that the descending noradrenergic system could "release" the spinal circuitry for stepping. These results were supported by work in our laboratory confirming that clonidine (intraperitoneally) can trigger hindlimb treadmill locomotion in adult chronic spinal cats (awake behaving animal) within the first week after spinalization (Barbeau et al. 1987). In a recent paper (Chau et al. 1998), we reported the effects of early locomotor training with daily injection of clonidine (intraperitoneally in 4 cats, and intrathecally in 1 cat) within the first week after spinal transection. In the present work, we have pursued these ideas with the aim of better identifying the potential of various noradrenergic drugs, in addition to clonidine, acting on different receptors to initiate and modulate locomotion.

In contrast to our previous work where drugs were injected intraperitoneally, the present study used an intrathecal cannula exclusively for drug delivery. This not only reduced some side effects encountered with intraperitoneal injections but also greatly expanded our ability to explore a wider variety of drugs. Because the drugs were injected directly into the intrathecal space of the spinal cord, central effects of the drugs dominated over peripheral effects. It also made possible testing drugs that do not cross the blood brain barrier, such as oxymetazoline and thus explore various types of alpha 2 agonists. Thus adult spinal cats implanted with an intrathecal cannula may serve as a unique model where the effects of different pharmacological agents on locomotion can be studied.

Although the importance of noradrenergic system in inducing and modulating locomotion in spinal animal was established, relatively little information is available on the specificity of the receptors involved in mediating these locomotor effects. Although both beta and alpha  noradrenergic receptors are present in the spinal cord (Nicolas et al. 1993; Timmermans and van Zwieten 1982), alpha  noradrenergic receptors have been shown in previous studies using DOPA or clonidine to be involved in triggering locomotion and thus would be the focus of this paper.

The alpha  noradrenergic receptors are subdivided broadly into the alpha 1 and alpha 2 subtypes. The two subtypes of noradrenergic receptors have been reported to mediate different functions. For example, it was found that activation of alpha 1 receptors facilitate the flexor reflex whereas activation of alpha 2 receptors appears to mediate inhibitory effects in acutely spinalized rats (Sakitama 1993). Clonidine acts primarily on the alpha 2-adrenergic receptor (Marshall 1983; Ruffolo and Hieble 1994; Timmermans and van Zwieten 1982). It was shown that clonidine could stimulate central norepinephrine receptors in acute spinal rats, suggesting the role of a alpha 2 receptor (Anden et al. 1970). So far, clonidine remained the noradrenergic agonist most widely used to induce, ameliorate and modulate locomotion in acute or chronic spinalized cats (Barbeau and Rossignol 1991; Forssberg and Grillner 1973; Rossignol et al. 1995). Little is known about the effects of other alpha 2-adrenergic receptors or effects mediated by alpha 1 adrenoceptors.

The purpose of this study was to explore the functional role of alpha 1 and alpha 2 adrenoceptors in the initiation and modulation of locomotion and cutaneous reflexes after spinal cord transection in chronic spinal cats. As spinalization removed all presynaptic receptors by removing all descending noradrenergic terminals, the receptors activated are presumed to be located postsynaptically. To compare with clonidine, other selective alpha 2 agonists, tizanidine and oxymetazoline were used, and yohimbine was injected in some cases to antagonize their effects. Methoxamine, a selective alpha 1-adrenoceptor agonist (Marks et al. 1990) and prazosin, a selective blocker also were studied. Finally, norepinephrine itself was injected.

A more detailed understanding of the noradrenergic drugs, and their actions mediated by different receptors, is important to enhance our ability to optimize the therapeutic use of drugs in patients with spinal cord injury and potentially better target the pharmacotherapy to offset more specific locomotor deficits.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Five adult cats were used for this study. They were trained to walk on a motor driven treadmill belt and could walk continuously for >= 15 min at 0.3-0.4 m/s. After training, they were chronically implanted with electromyographic (EMG) electrodes and an intrathecal catheter. In two cats, nerve cuffs electrodes also were chronically implanted on the superficial peroneal nerve. Once baseline recordings of the intact locomotion were made, cats were spinalized.

All surgeries were performed in aseptic conditions. Cats were anesthetized with intravenous pentobarbital (Somnotol, 35 mg/kg). Additional doses of barbiturates (3-5 mg/kg iv) were given as needed throughout the surgery to ensure that the animal remained deeply anesthetized. Lactate-Ringer solution was given continuously through an intravenous line during surgery. The body temperature was constantly monitored with a rectal thermometer and controlled by placing the cat on a heating pad. All procedures followed a protocol approved by the Ethics Committee of Université de Montréal.

INTRATHECAL CATHETERIZATION. The intrathecal cannulation technique was adapted from the procedure of Espey and Downie (1995). A length of Teflon tubing (24LW) was connected to a cannula connector pedestal (Plastic One) covered with a dust cap. The tip of the catheter was perforated with a few holes on the side to ensure drug infusion. Before insertion, the catheter was filled with sterile saline, and the dead space of the catheter was measured (~100 µl). With the cats's head secured in the stereotaxic frame, a midline incision was made from the cranium to C2-C3 level. One end of the catheter was secured on the skull with acrylic cement as a port of entry while the other end was inserted through an opening in the cisterna magna down to approximately L4-L5 (Fig. 1). In one spinal cat (CC4), X-rays were taken at different times after the injection of an radio opaque dye into the cannula and showed that the tip of the cannula was located at L5 and that the diffusion of the radio opaque material was localized within the lumbosacral region.


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  		FIG. 1. Scheme showing the experimental set-up during locomotion. Hindlimbs of the cat were placed on the moving treadmill belt while the forelimbs stood on a stationary platform (approx 2 cm above). Both the head connector and the cannula inlet port are fixed on the head as shown. Details of the recording procedures and synchronization procedures are described in METHODS. Four joint angles are measured so that flexion will result in a decrease of angular values. MTP, metatarso-phalangeal joint.

After the catheterization, the cannula was flushed daily with 100 µl sterile saline to prevent blocking. The location of the tip of the catheter was identified during postmortem examination and is listed for all cats in Table 1. Postmortem examination also revealed that the cannula can leave an imprint on the cord. This compression did not, however, produce any apparent locomotor deficits in our cats because they all walked well after the implantation.

 
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  		TABLE 1. All experimental trials

IMPLANTATION OF EMG ELECTRODES. A detailed description has been made elsewhere (Chau et al. 1998). Briefly, three cats were implanted with two 15-pin head connectors (TRW Electronic Components Group) while two cats were implanted with only one connector secured to the cranium using acrylic cement. Seven or 14 pairs of the Teflon-insulated stainless steel wires (previously soldered to the head connectors) were passed subcutaneously to small incisions made overlying the selected hindlimb muscles (Fig. 1). A pair of stainless steel wires was sewn into the belly of each muscle. Before insertion, a small portion of the Teflon coating was removed from the Teflon-insulated stainless steel wires to be inserted in the muscle. Unpaired wires from the last pin of each connector were placed under the skin of the neck to serve as an electrical ground. Bilaterally implanted muscles include iliopsoas (Ip), a hip flexor; sartorius (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; and tibialis anterior (TA), an ankle flexor. Electrodes also were inserted unilaterally into gluteus medius (Glu), a hip abductor and extensor, and in gastrocnemius medialis (GM), an ankle extensor and knee flexor.

IMPLANTATION OF NERVE CUFF ELECTRODES. In two cats (CC5 and CC7), bipolar cuff electrodes (Julien and Rossignol 1982) (~1 cm length) were used to stimulate the superficial peroneal nerve (~6 mm between electrodes leads). A 2-pin head connector, soldered with a pair of Teflon-insulated stainless steel wires was used. The stainless steel wires were led to the site of implantation subcutaneously. Using a custom-made apparatus, a U-shaped nerve cuff was made from polymer (Caulk Dentsply International). The wires were anchored to the nerve cuff and the small portion of stainless steel wires inside the cuff was cleared of the Teflon insulation. The superficial peroneal nerve was placed in the cuff followed by absorbable gelatin sponge (Sterispon) soaked with saline solution to prevent damages related to secondary swelling and was completely sealed off using polymer.

SPINALIZATION. A laminectomy was performed at the T13 vertebra. The dura was carefully removed, a few drops of xylocaine (2%) were placed on the spinal cord, and then a few injections (0.1-0.2 ml each) were made directly into the spinal cord at the level of transection area. The exact location of the intrathecal cannula first was identified to avoid causing any damage to the cannula, then the spinal cord was completely severed progressively using microscissors. The spinal canal could be visualized clearly, and 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-µm sections using the Kluver-Barrera method).

Postoperative cares

All animals were placed in an incubator immediately after surgery and monitored closely. Once the animals regained consciousness, they were placed in individual cages (104 × 76 × 94 cm) with food and water. Torbugesic (Butorphenol tartrate, 0.05 mg sc, every 6 h) was given in the first postoperative day to reduce discomfort. Spinal cats were placed in cages lined with foam mattresses and were attended to a few times daily to maintain the cleanliness of the head connectors, to flush the intrathecal cannula with sterile saline, to express the bladder manually, and to inspect and clean the hindquarters. All our spinal cats remained very healthy and were kept for a period of 2-9 mo (an averaged of 6 mo) after spinalization.

Recording procedures and protocol

A few days after the intrathecal catheterization and the implantation of EMG electrodes and/or nerve cuff electrodes, cats were placed on the treadmill to record locomotion. This served as the baseline controls (the intact trials). After spinalization, before drug injection (predrug trials), and at different intervals after each intrathecal drug injection (postdrug trials), locomotion and responses to mechanical and cutaneous stimulation were recorded.

Experiments were made at two stages after spinalization. The first was at the early stage (~1 wk) after spinalization when there was no spontaneous treadmill locomotion yet. These cats are referred to as early spinal cats. With time and training, spinal cats can attain a well-coordinated locomotor pattern with full weight support and plantar foot placement without drug injection (Barbeau and Rossignol 1987; Chau et al. 1998). These cats will be referred to as late-spinal cats.

Drug injections

The different noradrenergic drugs used in these experiments are the neurotransmitter norepinephrine (NE) [4-(2-amino-1-hydroxyethyl)-1,2-benzenediol] from RBI, alpha 1-agonist methoxamine [alpha -(1-aminoethyl)-2,5-dimethoxybenzenemethanol] from RBI, alpha 2 agonists including clonidine (2,6,-dichloro-N-2-imidazolidinylid-enebenzenamine) from Sigma, oxymetazoline {3-[(4,5-dihydro-1H-imidazol-2-yl)methyl]-6-(1,1-dimthylethyl)-2,4-dimethylphenol} from Sigma, and tizanidine [5-chloro-N-(4,5-dihydro-1H-imidazol-2-yl)-2,1,3-benzothiadiazol-4-amine] from Sandoz Pharmaceuticals. In two cats (CC6 and CC8), an alpha 2 antagonist, yohimbine [(16alpha ,17alpha )-17-Hydroxy yohimban-16-carboxylic acid methyl ester] from RBI and an alpha 1 antagonist, prazosin [1-(4-amino-6,7-dimethoxy-2-quinazo-linyl)-4-(2-furanylcarbonyl)piperazine] from Pfizer were used. The range of doses given during experiments was 4.9-12 mM for NE, 2.0-8.0 mM for methoxamine, 0.4-4.0 mM for clonidine, 1.7-3.4 mM for oxymetazoline, 1.0-3.9 mM for tizanidine, and 2.6 mM for both yohimbine and prazosin. All drugs were dissolved in sterile saline solution except prazosin, which was dissolved in 20% dimethyl sulfoxide, 40% distilled water, and 40% saline. Drugs were injected as a bolus into the spinal cord through the intrathecal cannula. Most bolus injections were of 100 µl, but sometimes cumulative doses were given with injection volumes ranging from 25 to 200 µl per dose. After each drug injection, a subsequent bolus injection of saline (~100 µl) was made to fill the dead space of the cannula and to ensure infusion of the drug into the intrathecal space of the spinal cord. The limit of volume given in one session was ~600 µl.

Locomotion

During the control period, locomotion at different speeds was recorded while the cats walked freely on the treadmill belt. After spinalization, the forelimbs of the spinal cat were placed on a platform (~2 cm above the treadmill) and locomotion of the hindlimbs was recorded (see Fig. 1). An acrylic plastic (Plexiglas) separator (not shown) was put between the hindlimbs to prevent crossing of the hindlimbs resulting from increased adductor tonus often seen in spinal cats. In the early period postspinalization, the experimenter lifted the tail of the cat to support the weight of the hindquarters of the cat and to provide equilibrium. With time, the cat could walk with complete weight support of the hindquarters, and the experimenter only held the tail to provide equilibrium of the hindquarters.

The EMG signals were amplified differentially (bandwidth of 100 Hz to 3 kHz). Twelve channels were recorded with a video recorder (Vetter Digital, model 4000A PCM recording adapter) with a frequency response of 1.2 kHz per channel.

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). For every recording session, 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 (see Fig. 1). Additional markers also were placed either on the treadmill frame or on the trunk of the cat for calibration (10 cm).

The kinematic and the EMG data were synchronized by means of a digital SMPTE (Society for Motion Picture and Television Engineers) time code. The time code was generated by a Skotel time code generator (model TCG-80N) and was recorded simultaneously on the EMG tape and on one audio channel of the VHS tape and was inserted as well into the video images.

Electrical stimulation

Single pulse of 250-µs 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.

Mechanical stimulation

Mechanical stimuli were delivered by tapping the dorsum of the paw with a custom-made hand-held tapper during the swing phase of locomotion. The tapper has a microswitch attached to indicate the moment of contact with the surface of the dorsum of the paw. The pulse generated by the switch was recorded on tape and also triggered a light-emitting diode (LED) recorded on the video tape. The stimulus was applied randomly during the swing phase of locomotion but not exceeding once every three step cycles.

Fast paw shake

To elicit a fast paw shake (FPS), the experimenter held the cat in the air and then dipped the paw into a bowl of lukewarm water. While both limb movements and EMG signals were recorded, only the EMG signal was analyzed for FPS.

Data analysis

Video images were digitized using two-dimensional PEAK Performance system (Peak Performance Technologies, Englewood, CA). Displacement data, encoded by the x and y coordinates of different joint markers, were measured at 60 fields/s (i.e., a temporal resolution of 16.7 ms). From these x-y coordinates, angular joint movements were calculated and could be displayed as continuous angular displacements (running averages of 5 values) for a normalized step cycle or as stick diagrams. Each stick figure was also displaced from the previous one by the distance traveled by the foot so that the horizontal axis is twice that of the vertical axis. The distance between stick figures is also proportional to the velocity of the movement.

EMG data during locomotion were played back on an electrostatic polygraph (Gould, Model ES 1000) and a typical record of the animal's performance before and after drug injection was selected for analysis. The EMG signals were digitized at 1 kHz. Using custom-made software, the onset and offset of bursts of activity were detected first automatically and then corrected manually if needed. The EMGs then were rectified and, using St as the onset of the cycle (occasionally Srt), the EMGs were averaged over a number of cycles. The duration and amplitude of the muscle bursts were measured from individual records. The mean amplitude was calculated as the integral of the rectified EMG burst divided by its duration.

EMG responses to the electrical stimulation was digitized at 1 kHz and computer averaged. Quantitative measures of the responses (amplitude and latency) were obtained using custom-made software which integrate the region that was consistently greater or less than the mean prestimulus period by 2 SD. For mechanical stimuli, the individual EMG responses to stimulation were shown before and after drug injection.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

The results reported here are from experiments in the five spinal cats in which different drugs were injected on different postspinal days as summarized in Table 1. All analyzed trials at different days are underlined. Even though some trials were not analyzed quantitatively, the videotapes and EMG data always were reviewed to verify the similarities or differences of drug effects in different spinal cats. Although a range of doses has been tested, the analyses reported here refer mainly to trials where a dose of 3-4 mM in a bolus of 100 µl was used for all alpha 1 and alpha 2 agonists; this seems to produce optimal locomotor effects. For the antagonists, doses of 2.5-2.6 mM were effective. In the case of NE, a higher dose, <= 12 mM, sometimes was required. The effects of a drug on locomotion were evaluated at two stages posttransection, at an early stage (early spinal) when there was no spontaneous locomotion (i.e., <8 days) and at a later stage when the locomotor pattern was already established (late spinal) before any drug injection. In all early spinal cats, usually within the first week posttransection, no well-organized sustained locomotion can be elicited before drug injection. The ability of the different noradrenergic agonists to initiate locomotion could then be tested.

Initiation of locomotion in early spinal cats

EFFECTS OF alpha 2-NORADRENERGIC AGONIST (CLONIDINE, TIZANIDINE, OXYMETAZOLINE). The ability of clonidine, a well-known alpha 2-noradrenergic agonist, to trigger locomotion was confirmed consistently here in four spinal cats and is shown in Fig. 2. In this 8-day (8d)-spinal cat (CC7), although there was no locomotion during the predrug trials (Fig. 2B), almost immediately (within 2 min) after clonidine injection (3.8 mM it injected as a bolus of 100 µl) a well-organized locomotor pattern was observed (Fig. 2C). There was a marked increase in stance and swing duration comparable to the intact locomotion (Fig. 2A) as shown in the stick figures. This remarkable quasi-instantaneous action of intrathecal clonidine also was seen in another spinal cat (CC4) in which locomotion was triggered within 3 min after injection. The clonidine-elicited locomotion can be characterized as readily triggered, requiring only a light touch to the perineum; adaptable to treadmill speeds <= 1.0 m/s; sustained, i.e., the cat could walk consistently 15-20 min at a time for a period of 2-3 h; and the effects last for ~5 h. Although the clonidine-elicited locomotion resembled the intact locomotion in many respects, there were also some distinct characteristics. For example, a knee sag often was observed toward the end of stance as noted in the stick figures [the iliac and hip markers are sloping downwards toward the end of the stance phase, something that is not normally seen before spinalization (Fig. 2A)]. Another distinct characteristic consistently observed after clonidine was a pronounced foot drag during the initial swing phase (Fig. 2C) that often was followed by a greater elevation of the foot at the end of swing before putting down the foot.


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  		FIG. 2. Effect of an alpha 2 agonist, clonidine, on the initiation of locomotion in an 8-day (8d) spinal cat (CC7). A: stick diagram (1-step cycle) and raw electromyographic (EMG) traces of hindlimb flexor and extensor muscles during intact locomotion before spinalization. Treadmill speed at 0.3 m/s. B: locomotion at 8d postspinalization before any drug injection. C: locomotion recorded at 2 min after clonidine injection (4 mM it). Muscle gains of ipsilateral (i) semitendinosus (St) and contralateral (co) St EMG were decreased to 0.4 and 0.5 times the gain of recording in intact cat.

Other alpha 2-noradrenergic agonists, tizanidine (n = 2) and oxymetazoline (n = 2), were also capable of initiating locomotion in early spinal cats within the first week posttransection. In Fig. 3, the stance, swing, and cycle duration as well as stance length (measured from kinematic values) in a 3d, 4d, and 8d spinal cat after injection of clonidine, oxymetazoline, and tizanidine, respectively, as well as a 8d spinal cat after injection of NE are shown. The values are expressed as a percentage of intact locomotion because the cats were not walking at this early stage posttransection. The three alpha 2 agonists triggered locomotion similarly by increasing the step cycle duration, especially the swing duration. The locomotion initiated by NE was not as good as that triggered by the alpha 2 agonists. For example, the stance length was 64% of the intact locomotion after NE as compared with the 99% after oxymetazoline injection. As shown in Table 2, although the cycle duration of the spinal locomotion was very small before clonidine injection at 3, 5, and 8d posttransection in cats CC4, CC5, and CC7, respectively, it was 113, 92, and 135% of intact values within minutes after clonidine injection. Similarly, after oxymetazoline injection in cat CC8 at 4d posttransection (3.4 mM it), the cycle duration was increased to 129% of intact locomotion. The ability of the cat to adapt its locomotion to increasing treadmill speed was also similar among the alpha 2 agonists. The maximum speed the early spinal cats can achieve after injection of tizanidine (n = 2), oxymetazoline (n = 1), and clonidine (n = 2) injection was 0.6-0.7, 0.8, and 0.9-1.0 m/s, respectively.


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  		FIG. 3. Histograms of cycle duration, stance duration, swing duration, and stance length (obtained form kinematic data) expressed as percentages of intact locomotion in 4 spinal cats: CC4 (3d), CC8 (4d), and CC6 (8d) and CC5 (8d) after intrathecal injection of clonidine (3.8 mM), oxymetazoline (3.4 mM), tizanidine (3.9 mM), and norepinephrine (12.0 mM), respectively. - - -, values obtained from the cats during intact locomotion before spinalization. Note that the cats were not walking before the drug injection.

 
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  		TABLE 2. Step cycle and muscle burst durations after the injection of noradrenergic drug in early spinal cats when no locomotion could be elicited before drug injection

The concomitant EMG changes also are listed in Table 2. After all three alpha 2 agonists, there was an increase in the knee flexor St activity, relatively more than that of the extensor activity that contributes to the marked increase in the swing duration. For example in cat CC4, after clonidine injection, ipsilateral (iSt) burst duration was 247% of intact, whereas the extensors, iGL and iVL, were 109 and 102% of intact. Similar results were observed in cat CC7 after clonidine injection. After tizanidine injection in cat CC6, the burst duration of iSt was also much more augmented (230% of intact) than that of the extensors iGL and iVL, which are 138 and 126% of intact. After injection of oxymetazoline, in cat CC8 the coSt burst duration (not shown) was also 319% of the intact locomotion as opposed to iGL and iVL, which are 95 and 122% of intact, respectively. In conclusion, alpha 2 agonists appeared to have a more potent effects on the hindlimb flexors muscles.

Although the three alpha 2 agonists were similar in their ability to trigger locomotion in early spinal cats, differences in the evoked locomotor pattern were seen. Although tizanidine resembled closely the effects of clonidine, the kinematics of the locomotor pattern triggered by oxymetazoline was different from that of clonidine. For example, the increase in hip flexion was more marked after oxymetazoline injection as compared with clonidine, and the corresponding hip joint angular excursion was 144 and 87% of intact, respectively. The foot drag was also much more exaggerated after clonidine injection than oxymetazoline. The ankle joint angular excursion of clonidine- and oxymetazoline-evoked locomotion were 197 and 137% of intact, respectively. The ability of the cat to support the weight of the hindquarter was good after oxymetazoline injection as compared with clonidine. For example, the knee sag, often observed after clonidine was not observed with oxymetazoline.

The time course of action among the three alpha 2 agonists was also different as shown in Fig. 4. The locomotor effects were evaluated by measuring the stance duration at a speed of 0.6 m/s. Within 5 min after clonidine or tizanidine injection, the cat could walk at 0.6 m/s (Fig. 4A). Oxymetazoline, on the other hand, had a much slower onset, taking hours instead of minutes to reach the maximal locomotor effects (Fig. 4B, note that the time scale is different from Fig. 4A). The cat could not walk at 0.6 m/s at 30 min or 2 h after injection; however, when recording was made on the next day, a marked increase in the locomotor ability could be seen. The duration of effects exerted by the three alpha 2 agonists was also different. After tizanidine injection, the cat could not walk at 0.6 m/s after 2.5 h, whereas after clonidine injection, the cat still could walk at this speed even at 4.5 h, an ability that only diminished at 6.5 h after injection. The effects of both clonidine and tizanidine completely disappeared on the following day. With oxymetazoline, however, locomotion at 0.6 m/s could be maintained for >= 2 days after drug injection. A marked reduction in locomotion was seen by the third day postinjection where the stance duration decreased by 40%. We do not, however, know the time it takes for the effects of oxymetazoline to completely wear off.


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  		FIG. 4. Time course of action of the 3 alpha 2-noradrenergic agonists. Changes in the stance duration (during locomotion at 0.6 m/s) as a function of time after the injection of clonidine, tizanidine, and oxymetazoline in cats CC4 (3d), CC7 (9d), and CC8 (4d), respectively, were measured to evaluate the effects of the drugs. Note the different time scale between A (clonidine and tizanidine) and B (oxymetazoline). In A, at 2.5 and 6.5 h after tizanidine and clondine injections, respectively, the stance duration was at 0 as the locomotion returned to the predrug nonwalking status. Effects of clonidine and tizanidine completely dissipated on the following day. In B, oxymetazoline took some 2 h to have an effect and lasted for several days.

The differences in the time course of action and locomotor pattern were consistently seen in all experiments, in all spinal cats both during early and late-spinal period (see Table 3).

 
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  		TABLE 3. Summary of drug effects on locomotion and cutaneous excitability

EFFECTS OF AN alpha 1-NORADRENERGIC AGONIST (METHOXAMINE). The ability of methoxamine to trigger locomotion in early spinal cats was much more inconsistent and different from the alpha 2-noradrenergic agonists. We have tested the effects of methoxamine in three early spinal cats, all of which had no locomotor activity before drug injection.

In two cats (CC5 and CC7), there was a significant increase in the ability of the cats to stand on a stationary surface, and to a varying degree, an increase in stepping movements after methoxamine injection. For example, in cat CC5, 90 min after methoxamine injection (Fig. 5B), there was an attempt to increase stepping, as seen in the EMG traces. In another cat (CC7), 15 min after methoxamine injection, although the increase in stepping ability was more pronounced (stance length was 88% of intact), it was never as convincing as that observed with alpha 2 agonists. For example, the cats could not walk consistently (>= 10 consecutive step cycles) with weight support of the hindquarters or walk beyond 0.2 m/s. In both spinal cats, however, transient bouts of nice locomotor activity (0.2 m/s) with good weight support and an increase in the amplitude of EMG activity in flexors and extensors could be triggered with time (Table 2). Figure 5C shows an example of bouts of locomotion (cat CC5) observed at 5.66 h after methoxamine injection. With strong perineal stimulation, this cat could walk with good weight support and plantar foot placement up to 0.2 m/s. The cycle duration and stance length increased to 87 and 76% of the intact, respectively (see Table 2). This was the maximal effects observed in this cat and is in sharp contrast with the effect of clonidine given to this cat 2 days later (5d posttransection; Fig. 5D). Sustained organized locomotion (0.4 m/s) with large steps, weight support, and foot placement was readily observed at 1.5 h postinjection of clonidine requiring only minimal perineal stimulation.


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  		FIG. 5. Effects of an alpha 1 agonist, methoxamine, on a spinal cat (CC5) at 3d posttransection as compared with the effects of clonidine (alpha 2 agonist) on the same cat at 5d posttransection. A: locomotion before methoxamine injection. B: 90 min after methoxamine injection the cat had rhythmic movements of the knee but very little movements of the hip. Hindlimb was being dragged on the treadmill with the paw behind the hip joint. C: 1 bout of locomotor activity at 0.2 m/s could be observed at a longer time interval after injection of methoxamine (5.66 h). Note that the EMG activity in the proximal muscles is still not well organized at least on the contralateral side. D: in contrast to the effects of methoxamine, 90 min after clonidine injection in the same spinal cat 2 days later (5d posttransection), there was a well-organized locomotion at a treadmill speed of 0.4 m/s characterized by large alternating steps and well-developed EMG activities of the hindlimbs even in the more proximal muscles such as sartorius (Srt) on both sides.

Thus from the observations of these two cats (CC5 and CC7), methoxamine appeared not to be as effective as the alpha 2 agonists in triggering locomotion. Despite an increase in stepping movements and bouts of organized locomotion with weight support, the cats never could walk beyond 0.2 m/s.

However, contrary to the above observations, methoxamine was found to be effective in triggering locomotion in another spinal cat (CC6) at 4 days posttransection as shown in Fig. 6. Before methoxamine injection, no walking could be triggered on the moving treadmill belt even with strong perineal stimulation (Fig. 6B). Three hours after injection (Fig. 6C), the locomotor pattern significantly improved and was robust, requiring only minimal perineal stimulation. The cat could walk with plantar foot placement, support the weight of the hindquarters, take large steps and adapt to treadmill speed <= 0.4 m/s. The step cycle duration and stance length at 0.4 m/s were 101 and 89% of intact, respectively. This locomotor ability persisted till the following day (5 days posttransection). Thus it appears that an alpha 1 agonist, methoxamine, was also capable of initiating locomotion at least in this cat at an early stage postspinalization.


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  		FIG. 6. alpha 1 agonist, methoxamine, initiated locomotion in a spinal cat, CC6, at 4d posttransection. A: locomotor pattern during intact condition at 0.4 m/s. B: no locomotion was seen before methoxamine injection. C: 3 h after methoxamine injection (4 mM it), organized locomotor pattern was recorded at the same treadmill speed as the intact locomotion. Alternating EMG bursts of activity were observed in the hindlimb muscles, whereas the hip flexor Srt of both hindlimbs showed more or less tonic activity.

In the same cat, 2 days later, injection of an alpha 1-noradrenergic antagonist, prazosin, was found to be effective in blocking the effects of methoxamine on locomotion as shown in Fig. 7. Within 30 min after injection, there was no plantar foot placement, and instead, the cat continually struck the treadmill with the dorsum of the paw and was no longer capable of supporting its weight during locomotion. The step cycle duration and stance length decreased to 45 and 29% of intact, respectively, as compared with the corresponding values of 105 and 118% of intact before prazosin injection. The effects of prazosin appears to wear off by 1.75 h after injection.


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  		FIG. 7. Locomotor effects of the alpha 1 agonist methoxamine as shown in the previous figure was blocked by an alpha 1 antagonist, prazosin. A: in spinal cat CC6, at 6d posttransection, no locomotion was seen before drug injection. B: 3 h after methoxamine injection (4 mM it), the cat could walk with weight support and plantar foot placement at a treadmill speed of 0.4 m/s. C: injection of prazosin markedly reduced the step amplitude 34 min after, and the rhythmic movements were confined to the knee and the ankle. Gain of coSt shown in B and C was increased 2.5 times relative to the predrug trials.

Thus it suggests that the effects on locomotion seen in this cat (CC6) could be attributed to the effects mediated by NE alpha 1 receptors.

DIFFERENCES IN LOCOMOTION INDUCED BY THE alpha 1 AND alpha 2 AGONISTS. The locomotor pattern triggered by the alpha 1-noradrenergic agonist, methoxamine, differed from that evoked by alpha 2-noradrenergic agonists such as clonidine. In the alpha 2-induced locomotion, an exaggerated foot drag at the onset of swing was a consistent observation (Figs. 2C and 5D); in contrast, in the alpha 1-induced locomotion, there was no foot drag at the onset of swing (Figs. 5C and 6C). In the methoxamine-induced locomotion, the weight support of the hindquarters was also much better than the clonidine-induced locomotion. This is reflected partially by the absence of knee sag in the methoxamine-induced locomotion (Figs. 5C and 6C) as compared with the clonidine-induced locomotion (Figs. 2C and 5D). As shown in the stick diagram (Fig. 6C), the iliac and hip trajectory remained leveled and no knee flexion was seen at the end of stance, both of which often were observed after clonidine injection (Figs. 2C and 5C). The extensor activity of hindlimb muscle was also much increased after methoxamine injection as compared with after clonidine injection. After methoxamine injection in cats CC5 and CC6, at 3d and 4d postspinalization, the VL amplitude were 150 and 190% of intact, and the GL amplitude were 161 and 163% of intact, respectively. After clonidine injection in cat CC4 at 3d postspinalization, the amplitude of VL and GL was 97 and 117% of intact, respectively. Also, in the clonidine-induced locomotion (Fig. 2C), the activity of proximal muscle such as the hip flexor, Srt, was well organized (Figs. 2C and 5D). In the methoxamine-induced locomotion, no organized Srt bursting activity can be seen at this stage but evolved with time (Figs. 6C and 8C).


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  		FIG. 8. Effect of norepinephrine (NE) on initiating locomotion in an 8d spinal cat. A: locomotion during intact condition. B: no locomotion was observed before drug injection. C: locomotor pattern at 1 h after the injection of NE (12 mM it). At a treadmill speed of 0.2 m/s, organized hindlimb EMG activity was seen. Note that the EMG activity of the hip flexor Srt is not well organized compared with knee flexors St or knee extensor vastus lateralis (VL).

The initial ability of the early spinal cats to follow the maximal treadmill speed was also different. After methoxamine injection, the maximum treadmill speed that cat CC6 (4d) could follow was only at 0.4 m/s as compared with the clonidine injection, where the maximum speed cats CC4 (3d) and CC8 (3d) could follow was 0.8 and 0.6 m/s, respectively. However, with time (6d posttransection), cat CC6 also could adapt to 0.6 m/s after methoxamine injection. Finally, the time course of actions are also different. The effects of the alpha 1 agonist methoxamine were much longer lasting as compared with the alpha 2 agonists clonidine and tizanidine with the exception of oxymetazoline, which also produced long-lasting effects.

Therefore, differential effects were observed with alpha 1 and alpha 2 agonists with respect to the locomotor pattern, EMG activity, the weight support ability and the time course of action.

EFFECTS OF NORADRENALINE. Noradrenaline also was capable of triggering locomotion in the early spinal cat (CC8). Figure 8 shows the locomotor pattern of the 8d-spinal cat (CC5) during intact, predrug, and postdrug period. There was no locomotion before drug injection (Fig. 8B). Locomotion with plantar foot placement began at 40 min after NE injection (not shown; 12 mM it). The pattern was transient, and with time it became more robust, and by 1 h, the cat could walk with plantar foot placement and weight support of the hindquarters (Fig. 8C). The raw EMG traces showed alternating bursting between the different hindlimb flexor and extensor muscles. The locomotion was characterized by steps shorter than in the intact. The step cycle duration and the stance length of the NE-triggered locomotion were 58 and 72% of the intact locomotion, respectively. Thus it appears that although NE readily triggered robust locomotion in early spinal cats similar to alpha 2 agonists, the effects were less potent than alpha 2 agonists as shown in Fig. 3. In addition to exerting partial alpha 2 effects, there was also no foot drag or knee sag observed in the NE-induced locomotion; this also resembled the effects observed with a alpha 1-noradrenergic agonist. It appears then that mixed alpha 1 and alpha 2 effects could be evoked by NE as could be expected.

Modulation of locomotion parameters in late-spinal cats

In late-spinal cats, when the cat was capable of spontaneous locomotion, the ability of these drugs to modulate the already established locomotor pattern and their effect on cutaneous reflex excitability was assessed. The cutaneous reflex excitability of the hindlimbs was assessed by the response to mechanical and electrical stimulation as well as FPS. The effects of the drugs on locomotion and cutaneous reflex in all spinal cats and different experimental trials are summarized semiquantitatively in Table 3.

MODULATORY EFFECTS OF alpha 2 AGONISTS. All three alpha 2 agonists, clonidine (12 injections), tizanidine (7), and oxymetazoline (7), could modulate the locomotor pattern in a similar fashion (Table 3). Figure 9 shows an example of the effects of tizanidine on locomotion in a 157d spinal cat (CC8). Before any drug injection, the locomotor pattern was well established with full weight support and plantar foot placement (Fig. 9A). Thirty minutes after the injection of tizanidine (cumulative dose 4.8 mM), there was a marked increase in the step length (117% of predrug) as shown in the stick figures (Fig. 9D), and an increase in the angular excursion in all joints, in particular the knee and ankle joint, as shown in the joint angle plots (Fig. 9E). An exaggerated foot drag at the onset of swing, resulting from an inadequacy to clear the ground during foot lift, also was observed as shown in the stick diagrams. The amplitude and duration of the flexor (Ip, Srt) muscles were increased; this may contribute to the increase in swing duration. The duration of the extensor (GM and VL) bursts also was increased, contributing to an increase in stance duration after tizanidine injection; however, the amplitude of the ankle extensors GM was decreased (Fig. 9F), which might explained partially the decrease in weight support of the hindquarters. The fact that these locomotor effects of tizanidine were mediated by alpha 2 adrenoceptors was further supported by the ability of yohimbine, an alpha 2-adrenoceptor antagonist, to block the effects (Fig. 9G). As soon as 15 min after yohimbine (2.5 mM it) injection, there was a decrease in step length. The cycle duration decreased by 10% of the predrug trial, the swing duration decreased by 20% of the predrug trials, the weight support increased with a corresponding increase in the ankle extensor GM activities (26% of the predrug trials), and the exaggerated foot drag disappeared.


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  		FIG. 9. Effects of an alpha 2 agonists, tizanidine, and an alpha 2 antagonist, yohimbine, on the locomotion of a spinal cat (CC8) at 157d posttransection. A-C: locomotion at treadmill speed of 0.4 m/s before receiving any drug. Duty cycles are represented by horizontal lines with downward arrows indicating foot contacts and upward arrows indicating foot lifts. D-F: locomotor pattern recorded 30 min after injection of a 3 mM dose of tizanidine after a 1st dose of 2 mM given 1.92 h before. G-I: locomotion recorded 15 min after yohimbine injected 27 min after the records in D-F.

Although the three alpha 2 agonists (clonidine, tizanidine, and oxymetazoline) modulated the cycle duration and step length of locomotion of late-spinal cats similarly, some differences were noted (see Table 3). For example, the weight support ability was more affected after clonidine injection as compared with oxymetazoline and tizanidine injection. The decrease in weight support ability was seen in 75% of trials tested with clonidine, whereas the weight support ability was largely unchanged after oxymetazoline injection. Three of seven trials (42.8%) after tizanidine reported a decrease in weight support ability. Also, the degree of side effects produced by these alpha 2 agonists in late-spinal cats were different. Both clonidine (3.8 mM it) and oxymetazoline (3.4 mM it) often produced some side effects (vomiting, dilated pupil, lethargy) but tizanidine never did (4.7 mM it). These observations were seen consistently in four spinal cats.

MODULATORY EFFECTS OF METHOXAMINE. Figure 10 shows an example in cat CC6 of a methoxamine injection alone (Fig. 10, D-F) followed by a superimposed injection of clonidine (Fig. 10, G-I), allowing us to describe the modulatory effects of the combination of an alpha 1 and an alpha 2 agonist.


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  		FIG. 10. Combined effects of a alpha 1 agonist, methoxamine, and alpha 2 agonist, clonidine, on an 11d spinal cat. A-C: stick diagrams, averaged angular plot and averaged normalized EMG data during locomotion before injection of any drug. D-F: locomotor pattern recorded 2.5 h after methoxamine injection alone. G-I: clonidine was injected 17 min after the previous recording, that is, 2.78 h after methoxamine injection. Locomotion recorded 10 min after clonidine (3.8 mM it) in the same cat during the same experiment.

After methoxamine injection (4 mM it), there was a marked increase in the extensor tonus of the hindlimbs and the joints appeared stiffer when the cat was standing (not shown). There was also some obvious spontaneous movements of the tail that were absent before. However, methoxamine did not significantly modulate the well-established locomotor pattern in late-spinal cats (11 injections; Table 3). As seen in the stick diagrams (Fig. 10D), there was no apparent increase in stance or swing duration as compared with the predrug trials. There was no foot drag nor knee sag at the end of stance (both are commonly seen with alpha 2 agonists). Also, there was little differences in the angular excursion before and after methoxamine injection (Fig. 10, B and E). There was, however, a marked increase in the EMG amplitude of the knee and ankle extensors, VL and GL (163 and 130%, respectively, of the predrug values), and the proximal hip extensor, Glu (increased fivefold). The burst duration of the Glu also was increased to 210% of the predrug value. The overall increase in the extensor muscle activity could contribute to the increased extensor tonus and resulted in a more rigid posture with extended hindlimbs.

Figure 10, G-I, shows the combined effects of methoxamine and clonidine on locomotion in the same cat. Clonidine was injected to the same cat within 2.78 h of the methoxamine injection, after marked effects of methoxamine were obtained. Ten minutes after clonidine injection, the stance and swing duration increased to 119 and 143%, respectively, of the preclonidine values (Fig. 10G). Although an exaggerated foot drag during the initial swing period was seen, there was no knee sag at the end of stance as often observed after clonidine. This may be related to the increased extensor tonus. In addition, burst duration of flexors such as St and coSt were increased to 282 and 176%, respectively, of the predrug value. The amplitude of the extensor muscle such as iVL and iGL, although slightly decreased as compared with after methoxamine injection, remained high at 131 and 100% of the predrug values, respectively. The proximal hip extensor, Glu burst amplitude and duration also remained high at 637 and 189% of the predrug value, respectively.

Thus the resultant locomotor pattern showed a summation of effects mediated by both alpha 1 and alpha 2 agonists.

MODULATORY EFFECTS OF NORADRENALINE. The NE-modulated locomotor pattern also resembled (6 injections) the combined effects of alpha 1 and the alpha 2 agonists (Fig. 11, J-L). Twenty-three minutes after NE injection (4.9 mM it) there was a significant increase in the stance and swing duration (Fig. 11J) similar to that seen with the alpha 2 agonist tizanidine (Fig. 11B) injected in the same spinal cat (CC7) at a different postspinal day. This is in contrast with the alpha 1 agonist methoxamine with which there are no significant changes in the stance and swing duration was seen (Figs. 11C and 10D). An exaggerated foot drag during initial swing also was observed in both tizanidine- and NE-induced locomotion but not in methoxamine-induced locomotion. Also similar to tizanidine-modulated locomotion (Figs. 9E and 11D), the angular excursions of all joints, in particular, the knee, ankle, and MTP joint, angular excursion were increased significantly after NE injection (Fig. 11K). These observations were consistently seen in three different spinal cats. Normalized EMG showed that after NE injection, the knee flexor St burst duration and amplitude increased to 139 and 143% of the predrug value, respectively, resembling the tizanidine-induced locomotion. On the other hand, the NE-modulated locomotion also resembled the alpha 1-modulated locomotion in some respects. For example, there was no knee sag at the end of stance (Figs. 10D and 11G). Also, the amplitude of the knee and ankle extensors increased to 287 and 229% of the predrug value, respectively (Fig. 11I), which was similar to the methoxamine-modulated locomotion (Fig. 10F).


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  		FIG. 11. Comparison of the effect of an alpha 2 and an alpha 1 agonist and NE in the same cat. Effects of alpha 2 agonist, tizanidine, alpha 1 agonist, methoxamine, and norepinephrine on a spinal cat CC7 at different posttransection days, respectively. A-C: locomotor pattern at 151d posttransection before any drug injection. D-F: on the same day (151d), locomotion recorded at 30 min after tizanidine injection. G-I: on 154d posttransection, locomotion recorded at 3 h after methoxamine injection. J-L: on the 164d posttransection, locomotor pattern recorded at 23 min after norepinephrine injection.

Figure 12 summarizes the percentage change of the step cycle, stance, and swing duration in all cats after injection of alpha 2 agonists, one alpha 1 agonist, and norepinephrine. Similar effects were observed in the three alpha 2 agonists (Fig. 12, A-C). The increase in step cycle duration ranges from 20 to 40% of the predrug trials and the increase in swing duration ranges from 30 to 80% of the predrug trials, whereas the increase in stance duration ranges from 10 to 40% of the predrug trials. Thus alpha 2 agonists increased the swing duration more than the stance duration. Similar to the alpha 2 agonist, the increase in swing duration after NE injection ranges from 116 to 166% of the predrug trials in three experiments done in spinal cats CC7 and CC8.


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  		FIG. 12. Histograms showing the modulatory effects of 3 alpha 2 agonists (clonidine, oxymetazoline, and tizanidine), the alpha 1 agonist methoxamine, and NE on the cycle, stance, and swing duration in different late spinal cats. The cycle, stance, and swing duration were expressed as percentages of the predrug trials.

In contrast, after methoxamine injection (Fig. 12D), there was very little changes in all three cats with respect to the step cycle duration, stance, and swing duration as compared with the alpha 2 agonists. However, the alpha 1 agonist methoxamine exerted marked effects on increasing the tonus and the weight support of the hindquarters of the cat, possibly by increasing the amplitude and duration of extensor muscles especially the proximal hip extensor such as Glu. The effects on locomotion after injection of NE resembled a combined effects of alpha 1 and alpha 2 agonists.

Modulation of cutaneous reflex excitability in late-spinal cats

In addition to changes observed in the locomotor pattern in cats after drug injection, there were also concurrent changes in the excitability of the cutaneous pathways as seen with mechanical or electrical stimulation and FPS. The results obtained from all spinal cats also are summarized in Table 3.

MECHANICAL STIMULATION. The alpha 2 agonists clonidine and oxymetazoline markedly reduced or abolished the response to tap in 100 and 67%, respectively, of all trials tested (Table 3). Tizanidine, also decreased the response to tap but to a lesser extent; the reflex amplitude was decreased in 50% of the trials but remained unchanged in 50% of the trial.

An example of the response to tap is shown in Fig. 13. Before clonidine (Fig. 13A), as soon as the tapper touched the paw there was a brisk response, i.e., a rapid knee, ankle, and MTP flexion, shown in the stick figures to clear the obstacle. Note that on the video records, the tapper was seen in contact with the dorsum of the paw in only one frame (2 fields) indicated by one arrow. After clonidine, the brisk response to tap also disappeared as previously reported (Barbeau et al. 1987). On contact with the tapper, the knee failed to flex; instead the paw pushed continuously onto the tapper and eventually, by inertia, the limb continued its trajectory.


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  		FIG. 13. Stick diagrams showing response to mechanical stimulation (tapper) applied to the dorsum of the paw during swing of cat CC4 (38d and 46d) and cat CC8 (27d) pre- and postclonidine, methoxamine, and NE injection, respectively. Arrows underneath the stick figures indicate the video frames where the dorsum of the paw was contacted with the tapper.

In contrast to the alpha 2 agonists, the alpha 1 agonist (11 injections) and NE (5) did not reduced the response to tap in any of the tested trials. As shown in Fig. 13B, in the same cat, CC4, after methoxamine, there were no marked changes in the swing duration, and the response to tap was still present. NE appeared to exert effects of both alpha 1 and alpha 2 types. As shown in Fig. 13C, there was both a marked increase in the swing duration (resembling the effects of clonidine) and the cutaneous reflex remained excitable (resembling the effects of methoxamine).

ELECTRICAL STIMULATION. Although alpha 2 agonists consistently (7 injections) increased the threshold of stimulation or decreased the reflex response to electrical stimulation, the alpha 1 agonist (4) and norepinephrine (6) reduced the threshold and increased the reflex amplitude (Table 3). Figure 14 shows an example of the activation of flexors and extensor muscles during the electrical stimulation of the superficial peroneal nerve before and after drug injection in the same cat, CC7, at rest (standing). After clonidine injection (Fig. 14A), there was also a marked decrease in the amplitude of the short latency response in the knee flexor St despite a much stronger stimulating current. Before clonidine, a current of 0.75 mA was sufficient to activate St. After clonidine, St was not activated even with a current as high as 3 mA, i.e., four times the strength used before clonidine injection.


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  		FIG. 14. Comparison of responses to electrical stimulation of the superficial peroneal nerve of cat CC7 at rest (standing) after different drugs. A: averaged response of 20 and 10 stimuli before and after clonidine injection, respectively. Current delivered before clonidine injection was 0.75 mA and was 3.0 mA after injection. No response can be seen even at this current. B: averaged response of 9 and 10 stimuli before and after methoxamine, respectively. Current of the stimulation stayed the same (0.6 mA) before and after methoxamine injection. C: averaged response to 10 and 15 stimuli before and after NE injection, respectively. Current of stimulation before and after NE injection was 0.5 mA.