Download Neurochemical excitation of propriospinal neurons facilitates

J Neurophysiol 105: 2818 –2829, 2011.
First published March 30, 2011; doi:10.1152/jn.00917.2010.
Neurochemical excitation of propriospinal neurons facilitates locomotor
command signal transmission in the lesioned spinal cord
Eugene Zaporozhets,1 Kristine C. Cowley,1 and Brian J. Schmidt1,2
1
Department of Physiology and 2Department of Internal Medicine, Section of Neurology, Faculty of Medicine, University
of Manitoba, Winnipeg, Manitoba Canada
Submitted 25 October 2010; accepted in final form 25 March 2011
locomotion; brain stem electrical stimulation; neonatal rat; in vitro
long direct bulbospinal projections,
reticulospinal axons in particular, activate locomotor circuitry
in the vertebrate spinal cord (for reviews see Grillner et al.
1997, Jordan et al. 2008). Using the in vitro neonatal rat brain
stem-spinal cord preparation, we showed that propriospinal
pathways also convey descending transmission of locomotor
command signals originating in the brain stem (Zaporozhets et
al. 2006). Reticulospinal and propriospinal systems are not
likely independent in this function. Rather, reticulospinal collaterals, which are known to terminate diffusely, both ipsilat-
IT IS WIDELY ACCEPTED THAT
Address for reprint requests and other correspondence: B. J. Schmidt, Dept.
of Physiology, Rm. 406, Basic Medical Sciences Bldg., Univ. of Manitoba,
745 Bannatyne Ave., Winnipeg, Manitoba, Canada R3E 0J9 (e-mail:
[email protected]).
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erally and contralaterally, in cervical, thoracic, and lumbar
segments (Jankowska et al. 2003; Matsuyama et al. 2004;
Peterson et al. 1975; Reed et al. 2008) are well-positioned
anatomically to distribute locomotor command signals to propriospinal neurons throughout the rostrocaudal extent of the
spinal cord. During electrical stimulation of the brain stem,
propriospinal transmission alone, independent of long direct
projections, was sufficient to activate locomotor-like activity in
27% of in vitro rat preparations (Cowley et al. 2008). Thus
regeneration of propriospinal connections and artificial enhancement of signal propagation through residual intact propriospinal pathways, even without regrowth of long direct
brain stem projections to the lumbar cord, are attractive strategies to pursue for restoring spinal cord function after injury.
Evidence of propriospinal system plasticity participating in
the formation of functional bypass circuits in the lesioned cervical
cord has been shown in rat (Bareyre et al. 2004) and cat preparations (Fenrich and Rose 2009). Propriospinal mechanisms also
contribute to motor recovery after spinal cord injury in the lamprey (McClellan 1994), chick embryo (Sholomenko and Delaney
1998), and rat (Arvanian et al. 2006a, 2006b). Courtine et al.
(2008) demonstrated that recovery of hindlimb stepping in mice
with T7 and contralateral T12 hemisections was associated with
an increased number of propriospinal neurons in the interlesion
zone. Murray et al. (2010) also recently showed that rats with T6
and contralateral T12 hemisections improve their hindlimb locomotor scores over a period of several weeks.
The present study of lesioned spinal cord preparations examines whether neurochemical excitation focused specifically
on thoracic propriospinal neurons (i.e., located rostral to
hindlimb locomotor circuitry) facilitates descending propagation of the supraspinal command signal. This approach contrasts with previous studies that involved neurochemical activation of locomotor central pattern-generating (CPG) circuitry
in the lumbar segments through systemic administration of
drugs or local application to the lumbar region. For instance, in
vivo studies have shown that hindlimb stepping is promoted by
L-3,4-dihydroxyphenylalanine (L-DOPA) (Budakova 1971;
Grillner and Zangger 1979; Jankowska et al. 1967a, 1967b),
clonidine (Barbeau et al. 1987; Marcoux and Rossignol 2000;
Forssberg and Grillner 1973), serotonergic agents (Antri et al.
2002, 2003, 2005; Barbeau and Rossignol 1990, 1991; Feraboli-Lohnherr et al. 1999; Fong et al. 2005; Guertin 2009;
Hayashi et al. 2010; Ichiyama et al. 2008; Kim et al. 1999,
2001; McEwen et al. 1997; Viala and Buser 1971), and
excitatory amino acid receptor agonists (Chau et al. 2002;
Douglas et al. 1993; Giroux et al. 2003). Similarly, numerous
reports using in vitro vertebrate preparations describe direct
0022-3077/11 Copyright © 2011 the American Physiological Society
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Zaporozhets E, Cowley KC, Schmidt BJ. Neurochemical excitation
of propriospinal neurons facilitates locomotor command signal transmission in the lesioned spinal cord. J Neurophysiol 105: 2818 –2829, 2011.
First published March 30, 2011; doi:10.1152/jn.00917.2010.—Previous
studies of the in vitro neonatal rat brain stem-spinal cord showed that
propriospinal relays contribute to descending transmission of a supraspinal command signal that is capable of activating locomotion.
Using the same preparation, the present series examines whether
enhanced excitation of thoracic propriospinal neurons facilitates propagation of the locomotor command signal in the lesioned spinal cord.
First, we identified neurotransmitters contributing to normal endogenous propriospinal transmission of the locomotor command signal by
testing the effect of receptor antagonists applied to cervicothoracic
segments during brain stem-induced locomotor-like activity. Spinal
cords were either intact or contained staggered bilateral hemisections
located at right T1/T2 and left T10/T11 junctions designed to abolish
direct long-projecting bulbospinal axons. Serotonergic, noradrenergic,
dopaminergic, and glutamatergic, but not cholinergic, receptor antagonists blocked locomotor-like activity. Approximately 73% of preparations with staggered bilateral hemisections failed to generate locomotor-like activity in response to electrical stimulation of the brain
stem alone; such preparations were used to test the effect of neuroactive substances applied to thoracic segments (bath barriers placed at
T3 and T9) during brain stem stimulation. The percentage of preparations developing locomotor-like activity was as follows: 5-HT
(43%), 5-HT/N-methyl-D-aspartate (NMDA; 33%), quipazine (42%),
8-hydroxy-2-(di-n-propylamino)tetralin (20%), methoxamine (45%),
and elevated bath K⫹ concentration (29%). Combined norepinephrine
and dopamine increased the success rate (67%) compared with the use
of either agent alone (4 and 7%, respectively). NMDA, Mg2⫹ ion
removal, clonidine, and acetylcholine were ineffective. The results
provide proof of principle that artificial excitation of thoracic propriospinal neurons can improve supraspinal control over hindlimb locomotor networks in the lesioned spinal cord.
FACILITATION OF LOCOMOTOR COMMAND SIGNAL TRANSMISSION
J Neurophysiol • VOL
data have been presented previously in abstract form (Cowley
et al. 2009).
METHODS
Research protocols used in this study were in compliance with the
Canadian Council on Animal Care and were approved by the University of Manitoba Animal Protocol Review Committee. SpragueDawley rats (1–5 days old) were anesthetized with isoflurane, decerebrated at the midcollicular level, eviscerated, and placed in a bath
chamber containing artificial cerebrospinal fluid (ACSF) composed as
follows (in mM): 128 NaCl, 4.0 KCl, 0.5 NaH2PO4, 1.5 CaCl2, 21
NaHCO3, 1.0 MgSO4, and 30 glucose, equilibrated to pH 7.4 with
95% O2-5% CO2. The brain stem and spinal cord were then isolated
ventral side up and bilaterally intact. Experiments were conducted at
room temperature (ACSF ⬃22°C). Preparations were left in the bath
solution, unstimulated, for ⬃1h before we attempted to elicit locomotor-like activity via electrical stimulation of the brain stem.
Ventral root recordings, obtained using glass suction electrodes,
were band-pass filtered (30 –3,000 Hz), digitized, and captured using
Axoscope software (version 9.0; Axon Instruments). Axoscope files
were converted to an appropriate binary format for further analysis
with the use of special purpose software (developed by the Spinal
Cord Research Centre, University of Manitoba).
In some preparations, hemisections were made in the rostral (right
T1/T2) and contralateral caudal (left T10/T11) thoracic regions. The
bath was then partitioned using thin plastic barriers sealed at cord
contact edges with petroleum jelly. Barriers were placed such that
spinal neurons in the interlesion zone (T3 through T9 inclusive) could
be selectively exposed to neurochemicals. As noted in RESULTS, intact
spinal cords were used in some experiments, with barriers placed at
C1 and T8/T9 for application of neurochemicals to the cervicothoracic
region or at T11 for selective application of neurochemicals caudal to
this level. Excitatory neurochemicals were applied at concentrations
subthreshold for evoking locomotor-like activity in the absence of
brain stem stimulation. All neurochemical concentrations refer to final
bath concentrations.
Electrical stimulation of the brain stem was performed as previously described (Zaporozhets et al. 2004). In brief, an ACSF-filled
glass electrode, with a tip diameter of 200 –300 ␮m, was placed in
contact with the ventral surface of the brain stem. Bipolar stimulation
was used to deliver monophasic rectangular current pulses (4 –20 ms,
0.5–5 mA, 0.8 –2.0 Hz). Stimulation was applied for a maximum of
2–3 min per test episode. For experiments involving application of
neurotransmitter antagonists to thoracic segments in an effort to
abolish brain stem-evoked locomotor activity, baseline rhythmic activity in the absence of drug application was first established. The
selected antagonist was then applied using a range of concentrations
as needed, using the baseline brain stem stimulation parameters. If
locomotor-like activity was abolished, higher stimulation current was
administered in an effort to determine whether locomotor-like activity
could break through. The antagonist was considered capable of
blocking propriospinal transmission only if it blocked brain stemevoked locomotor-like activity at all stimulation strengths. For experiments involving neurochemical excitation of thoracic propriospinal
neurons in an effort to facilitate brain stem-evoked locomotor activity,
we first established that locomotor-like activity could not be elicited
regardless of brain stem stimulation intensity. Excitatory neurochemicals were then applied using a range of concentrations as needed while
the brain stem was stimulated at a range of current strengths (from 0.5
to 5 mA).
Criteria used to classify lumbar ventral root discharge as locomotor-like are in accordance with our previous work (e.g., Cowley et al.
2008). In particular, the ventral root discharge pattern was deemed
locomotor-like if 1) alternation was observed between the left and
right sides at L2 and/or between left and right sides at the L5 level,
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neurochemical stimulation of locomotor CPG circuitry using
excitatory amino acid, monoaminergic, and cholinergic agonists (Barry and O’Donovan 1987; Cazalets et al. 1992; Cohen
and Wallen 1980; Cowley and Schmidt 1994, 1997; Dale and
Roberts 1984; Gabbay and Lev-Tov 2004; Grillner et al. 1981;
Guertin and Hounsgaard 1998; Harris-Warrick and Cohen
1985; Jiang et al. 1999; Jovanovic et al. 1996; Kiehn and
Kjaerulff 1996; Kremer and Lev-Tov 1997; Kudo and Yamada
1987; Madriaga et al. 2004; McDiarmid et al. 1997; McLean
and Sillar 2003; Panchin et al. 1991; Poon 1980; Smith and
Feldman 1987; Sqalli-Houssaini and Cazalets 2000; Whelan et
al. 2000). Cyproheptadine and clonidine promote stepping in
humans with spinal cord injury (Fung et al. 1990; Remy-Neris
et al. 1999; Stewart et al. 1991; Wainberg et al. 1990).
Systemic administration of drugs in vivo or whole cord applications of drugs in vitro may influence locomotor-related
cervicothoracic propriospinal neurons as well as lumbar CPG
circuitry.
Relatively little information is available as to which neurotransmitters may be involved in locomotor-related propriospinal transmission. However, assuming the locomotor command signal input to propriospinal neurons is delivered by
reticulospinal projections, monoaminergic and/or glutamatergic mechanisms likely participate. A role for serotonin is also
suggested by the observation that serotonin application to the
lumbar cord will not elicit lumbar locomotor-like activity
unless serotonin is also used to excite neurons in the cervicothoracic region (Cowley and Schmidt 1997). Similarly, combined serotonin/N-methyl-D-aspartate (NMDA) excitation of
neurons in the cervical enlargement (Ballion et al. 2001;
Cowley and Schmidt 1997) or rostral cervical segments (Cowley et al. 2008) can induce locomotor-like activity in the
hindlimbs. Electrical stimulation of serotonin-containing reticulospinal neurons in the neonatal rat parapyramidal region
evokes locomotion (Liu and Jordan 2005). In favor of a role for
noradrenergic mechanisms is the observation that intraspinal
injection of the ␣2-noradrenergic receptor antagonist yohimbine into pre-CPG segments in the lower thoracic and upper
lumbar region of the cat blocks spontaneous locomotion (Delivet-Mongrain et al. 2008). Because of the ubiquitous nature
of excitatory amino acid transmission in the nervous system, it
seems reasonable to speculate that glutamate activates propriospinal neurons and is released by them. Thus monoaminergic
and excitatory amino acid receptors on cervical and/or thoracic
propriospinal neurons are logical targets for artificial stimulation in an effort to facilitate transmission of the locomotor
command signal in the lesioned spinal cord.
The main goal of the present study was to determine whether
neurochemically enhanced propriospinal transmission in the
thoracic region improves supraspinal control over hindlimb
locomotor circuitry located in the lumbar cord. We first
screened a variety of receptor antagonists to determine which
neurotransmitter systems contribute to locomotor-related propriospinal transmission in response to electrical stimulation of
the brain stem in the neonatal rat preparation. We then investigated the effect of neurochemically enhanced propriospinal
excitation on locomotor command signal propagation using
preparations with staggered bilateral hemisections in the thoracic region. The latter preparations were selected on the basis
of failing to produce lumbar locomotor-like activity in response to brain stem stimulation alone. Some of the following
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FACILITATION OF LOCOMOTOR COMMAND SIGNAL TRANSMISSION
and 2) ipsilateral alternation was present between L2 (predominantly
flexor-related activity) and L5 (predominantly extensor-related activity) on at least one side. In some experiments, T8 ventral root activity
was monitored instead of L5, which allowed monitoring of activity in
the interlesion zone as well as the lumbar (L2) cord. However, in these
preparations without L5 recordings, we refer only to rhythmic activity, based on alternating left-right L2 discharge, rather than locomotor-like patterns, because full criteria could not be assessed.
RESULTS
Effect of Neurotransmitter Antagonists on Propriospinal
Transmission in Preparations Producing Lumbar
Locomotor-Like Activity During Brain Stem
Stimulation Alone
Preparations with intact spinal cords. The nonselective
5-hydroxytryptamine (5-HT) receptor antagonist mianserin
(50 –75 ␮M) was applied to the C1–T8 bath compartment
while the brain stem was electrically stimulated. Lumbar root
locomotor-like discharge was abolished in all preparations
(n ⫽ 4/4; Fig. 1). Another nonselective 5-HT receptor antagonist,
ketanserin (100 ␮M), had the same effect (n ⫽ 1/1). Of note,
neither of these antagonists is entirely specific for 5-HT receptors, because they also bind to ␣-noradrenergic and histamine
receptors (Hoyer et al. 1994).
The dopamine receptor antagonist haloperidol (10 –75 ␮M)
blocked brain stem-evoked locomotor-like activity in the lumbar region when applied to the C1–T8 region of the intact
spinal cord (n ⫽ 6/6; Fig. 2A). When haloperidol (15–20 ␮M)
was applied caudal to T11, brain stem-evoked locomotor-like
activity was also abolished (n ⫽ 2/2; Fig. 2B). Thus dopaminergic receptor activation appears to contribute to cervicothoracic propriospinal signal propagation and to activation of
locomotor circuitry in the hindlimb segments.
Application of the ␣1,2-noradrenergic receptor antagonist
prazosin (65 ␮M, n ⫽ 2/2) or the ␣2-noradrenergic receptor
antagonist yohimbine (5 ␮M, n ⫽ 6/6) to the C1–T8 region
suppressed lumbar locomotor-like activity in response to brain
stem stimulation (Figs. 3A and 4A, respectively). Similarly,
both prazosin (65 ␮M, n ⫽ 2/2) and yohimbine (15 ␮M, n ⫽
1/2) blocked brain stem-induced rhythmic activity when applied on locomotor circuitry located caudal to T11 (Figs. 3B
and 4B, respectively).
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Fig. 1. Effect of 5-HT receptor blockade in the cervicothoracic region on brain
stem-evoked rhythmic activity. A1: a locomotor-like pattern of lumbar ventral
root discharge was evoked by electrical stimulation of the brain stem.
A2: application of mianserin to the C1–T8 bath compartment blocked rhythmic
activity. Note that for this and subsequent data, regularly occurring spikes are
artifacts due to brain stem electrical stimuli. Superimposed artifacts related to
brief high-frequency trains of stimulation are also noted, as shown 6 times in
A2. The high-frequency trains were applied in an attempt to induce rhythmic
activity in preparations unresponsive to ongoing low-frequency stimulation. L,
left; R, right, N-ACSF, normal artificial cerebrospinal fluid.
Preparations with staggered bilateral hemisections. A theoretical limitation of the intact brain stem-spinal cord preparation is that an inhibitory influence of receptor antagonists on
propriospinal synaptic activity may be obscured by preserved
transmission through bulbospinal axons projecting directly to
lumbar locomotor circuitry. However, in the present series this
may not have been a major factor because, as noted above,
receptor antagonists with actions on all three monoamine
systems tested (dopaminergic, noradrenergic, and serotonergic)
suppressed brain stem-induced lumbar locomotor-like activity.
Nonetheless, this series included experiments using preparations with staggered contralateral hemisections made at the
right T1/T2 and left T10/T11 junctions. These lesions abolish
all direct-projecting bulbospinal input to the lumbar cord
(Cowley et al. 2008). Although propriospinal relays are also
disrupted, at least partially, by these lesions, any brain stem
signal reaching the lumbar region in such preparations must do
so via at least one synaptic relay and cross projection. Thus this
model enables investigation of the effects of neurochemical
manipulation of propriospinal transmission independent of the
influence of long direct projections to the lumbar cord.
As was the case for intact spinal cord preparations, monoamine receptor antagonists (applied to the thoracic interlesion
zone at T3–T9) blocked brain stem-induced locomotor-like
activity in the lumbar region. More specifically, rhythmic
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The results are divided into two main sections. First, preparations with either intact or lesioned spinal cords, capable of
generating lumbar locomotor-like activity in response to brain
stem stimulation, were used to systematically screen the suppressive influence of a variety of neurotransmitter antagonists.
This survey was used to determine which endogenous neurochemicals normally contribute to propriospinal transmission of
the locomotor command signal. The second section used lesioned spinal cord preparations (staggered contralateral hemisections) that failed to produce lumbar locomotor-like activity in response to brain stem stimulation alone. Approximately 73% of such preparations fail to display locomotion
(Cowley et al. 2008). A variety of excitatory agents were thus
applied to the thoracic cord to determine whether enhanced
propriospinal excitation enabled the emergence of locomotor
activity in the lumbar region when combined with brain stem
stimulation.
FACILITATION OF LOCOMOTOR COMMAND SIGNAL TRANSMISSION
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Effect of Neurochemical Excitation of Propriospinal Neurons
on Locomotor Command Signal Transmission in Lesioned
Preparations Unresponsive to Brain Stem Stimulation Alone
The effect of neurochemical agents and manipulations was
examined in preparations unresponsive to brain stem stimulation alone. Given the results using receptor antagonists, we
were particularly interested in the potential of serotonergic,
dopaminergic, noradrenergic, and glutamatergic agonists to
facilitate propagation of the locomotor signal. In these experiments, subthreshold concentrations of excitatory agents were
applied to the thoracic cord. That is, although neurochemical
application to the cervical and/or thoracic regions can sometimes induce rhythmic activity in the lumbar region (Ballion et
al. 2001; Cowley et al. 1997, 2008), in the present experiments
neurochemicals were applied at concentrations confirmed in
each preparation to be subthreshold for induction of rhythmic
activity in the absence of brain stem stimulation. The following
results are summarized in Table 1.
During brain stem stimulation, bath application of 5-HT
(10 –50 ␮M) to the thoracic region (T3–T9) of preparations with
staggered bilateral hemisections, at the right T1/T2 and left T10/
T11 junctions, enabled the emergence of locomotor-like activity
in the lumbar region in 13 of 30 preparations (Fig. 6A). In three
Fig. 2. Effect of dopamine receptor blockade in the cervicothoracic and caudal
spinal cord regions on brain stem-evoked rhythmic activity. A1: a locomotorlike pattern of lumbar ventral root discharge was evoked by electrical stimulation of the brain stem. A2: application of haloperidol to the C1–T8 bath
compartment blocked rhythmic activity, even in response to 4 attempts using
high-frequency trains of stimulation. B1: in another preparation, a locomotorlike pattern of ventral root discharge was evoked by electrical stimulation of
the brain stem. B2: application of haloperidol caudal to the T11 level suppressed brain stem-induced activity. B3: rhythmic activity reappeared in
response to brain stem stimulation after washout of haloperidol.
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activity was suppressed by the 5-HT antagonist ketanserin
(50 –75 ␮M, n ⫽ 3/7), the dopamine antagonist haloperidol
(10 – 80 ␮M, n ⫽ 6/9, Fig. 5), and the ␣1,2-noradrenergic
antagonist prazosin (35– 65 ␮M, n ⫽ 4/7). On the other hand,
SB269970 (10 – 40 ␮M), a serotonin antagonist that has relatively greater selectivity for 5-HT7 receptors, failed to suppress
locomotor-like activity (n ⫽ 3/3). Clozapine (1–30 ␮M), a
nonspecific antagonist with high affinity for 5-HT7 receptors as
well as actions on other 5-HT and ␣-noradrenergic receptors
(Svensson 2003), blocked brain stem-evoked locomotion in six
of nine preparations. In the example shown in Fig. 5A, thoracic
(T8) ventral roots were recorded along with L2 ventral roots.
Alternating lumbar ventral root activity was restored after
haloperidol washout, whereas T8 recordings remained silent
(Fig. 5A3). The reason for this is not known, but it may reflect
a greater capacity of the upper lumbar segments to generate
rhythmic activity in response to brain stem stimulation, compared with thoracic segments, in the presence of incomplete
washout of haloperidol.
The NMDA receptor antagonist AP-5 (20 – 80 ␮M) blocked
locomotion in four of four preparations. Acetylcholine receptor
blockade using atropine failed to abolish locomotor-like activity
in the three preparations tested, consistent with earlier observations of this antagonist when applied to the cervicothoracic region
of nonlesioned preparations induced using chemical or electrical
stimulation of the brain stem (Zaporozhets et al. 2006).
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additional preparations with staggered lesions, brain stem stimulation evoked rhythmic activity in one or two roots in the absence
of 5-HT application; subsequent 5-HT application to the thoracic
region increased the number of rhythmically active roots. Similarly the 5-HT receptor agonist quipazine (10 ␮M) promoted
lumbar locomotor-like activity in response to brain stem stimulation in three of seven preparations that initially failed to develop
rhythmic activity in response to brain stem stimulation alone.
Application of the 5-HT1A receptor agonist 8-hydroxy-2-(di-npropylamino)tetralin (8-OH-DPAT; 1–30 ␮M) to the thoracic
cord facilitated locomotor-like discharge in only 2 of 10 preparations that failed to display locomotion in response to brain stem
stimulation alone.
The effectiveness of brain stem stimulation depended on
both the stimulus intensity and concentration of 5-HT. Figure
6B shows results using 5-HT at 10, 30, and 50 ␮M in seven
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preparations, at four different strengths of brain stem stimulation. At a stimulation intensity of 1 mA, only the high concentration of 5-HT (50 ␮M) facilitated locomotor-like activity
(n ⫽ 1/7). At a stimulation strength of 2 mA, 30 and 50 ␮M 5-HT
were effective (n ⫽ 2/7 and 4/7, respectively). At higher brain
stem stimulus intensities (ⱖ3 mA), 10 ␮M 5-HT was effective
(n ⫽ 3/7) in preparations otherwise unresponsive to the same
intensity of brain stem stimulation in the absence of 5-HT.
However, even at the maximum stimulation strength (4 mA),
higher concentrations of 5-HT (30 and 50 ␮M) were effective
in more preparations (n ⫽ 4/7 and 5/7, respectively).
The combination of 5-HT (10 –50 ␮M) and NMDA (2–5
␮M) promoted locomotor-like activity in response to brain
stem stimulation in three of nine preparations unresponsive to
either brain stem stimulation or neurochemical application
(T3–T9) alone. It seems, however, that the facilitatory effect on
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Fig. 3. Effect of ␣-noradrenergic receptor blockade, using prazosin, in the cervicothoracic and caudal cord regions on brain stem-evoked rhythmic activity.
A1: a locomotor-like pattern of lumbar ventral root discharge was evoked by electrical stimulation of the brain stem. A2: application of prazosin to the C1–T8
bath compartment blocked rhythmic activity, except for a few bursts in response to high-frequency trains of brain stem stimulation. B1: in another preparation,
locomotor-like rhythm was induced by electrical stimulation of the brain stem. B2: application of prazosin caudal to the T11 level suppressed rhythmic discharge.
B3: locomotor-like activity reemerged in response to brain stem stimulation after washout of prazosin.
FACILITATION OF LOCOMOTOR COMMAND SIGNAL TRANSMISSION
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thoracic propriospinal transmission was mainly related to 5-HT
actions, because NMDA alone (2– 4 ␮M) uniformly failed to
enable locomotor-like activity (n ⫽ 0/14; Fig. 7, A1 and A2),
despite the fact that the NMDA antagonist AP-5 consistently
blocked locomotor-like activity evoked by brain stem stimulation (n ⫽ 4/4). In addition, the percentage success rate using
5-HT/NMDA (33%) was less than that using 5-HT alone
(43%). In nine preparations unresponsive to brain stem stimulation alone, an attempt was made to enhance NMDA receptor channel conductance, in the thoracic cord, by using Mg2⫹free bath solution (Mayer and Westbrook 1987). This approach
also failed to facilitate propriospinal transmission of the locomotor command signal.
Norepinephrine application to the thoracic cord facilitated
brain stem-evoked hindlimb rhythmic activity in only 1 of the
28 preparations tested. On the other hand, the noradrenergic
receptor ␣1-agonist methoxamine (40 –100 ␮M) promoted
rhythmic activity in 5 of 11 preparations that otherwise failed
to develop rhythmic activity in response to brain stem stimulation alone (Fig. 7A3). The ␣2-noradrenergic agonist clonidine
(30 – 60 ␮M) was ineffective in all five preparations tested.
Dopaminergic (100 –500 ␮M) stimulation of thoracic neurons promoted rhythmic discharge in response to brain stem
stimulation in 3 of 15 animals. However, the pattern was
locomotor-like in only one of these preparations. Interestingly,
the combination of dopamine and norepinephrine more effectively facilitated locomotor-like output (n ⫽ 6/9; Fig. 8) than
either agent applied individually, suggesting a synergistic action.
Consistent with the failure of atropine to suppress brain
stem-induced lumbar locomotor-like activity, muscarinic reJ Neurophysiol • VOL
ceptor activation using acetylcholine (20 –50 ␮M) combined
with the acetylcholinesterase inhibitor edrophonium (100 ␮M)
consistently failed to facilitate rhythmic output in response to
brain stem stimulation (n ⫽ 0/11).
In seven preparations, the excitability of neurons was elevated in a nonspecific fashion by raising the concentration of
K⫹ ions in the thoracic bath solution to 7–9 mM. This promoted lumbar locomotor-like activity in two preparations that
otherwise failed to produce rhythmic activity during brain stem
stimulation alone.
DISCUSSION
A major finding of this study is that during brain stem
stimulation, neurochemical excitation of propriospinal neurons
located in the thoracic region facilitates the production of
lumbar locomotor-like activity in lesioned preparations that fail
to respond to brain stem stimulation alone. Because bathapplied receptor agonists and antagonists do not influence
axons of passage, the results further support the concept that
propriospinal relays contribute to propagation of the descending locomotor command signal (Cowley et al. 2008; Zaporozhets et al. 2006) in addition to long direct reticulospinal
pathways.
The results of the first series of experiments, using receptor
antagonists, suggest multiple neurotransmitter systems, including glutamatergic, serotonergic, dopaminergic, and noradrenergic, participate in locomotor-related propriospinal transmission. This finding is not surprising, considering numerous
studies, using a variety of vertebrate preparations, have indicated that direct activation of locomotor circuitry isolated
below a spinal cord transection can be achieved using a variety
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Fig. 4. Effect of ␣-noradrenergic receptor blockade, using yohimbine, in the cervicothoracic and caudal cord regions on brain stem-evoked rhythmic activity.
A1: a locomotor-like pattern of lumbar ventral root discharge was evoked by electrical stimulation of the brain stem. A2: application of yohimbine to the C1–T8
bath compartment blocked rhythmic activity. B1: in another preparation, locomotor-like rhythm was induced by electrical stimulation of the brain stem.
B2: application of yohimbine caudal to the T11 level suppressed the rhythmic discharge.
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Table 1. Summary of the effectiveness of neurochemical
excitation of propriospinal neurons in T3–T9 segments on the
capacity to generate lumbar locomotor-like activity during brain
stem stimulation
Fig. 5. Effect of dopaminergic receptor blockade on thoracic cord segments
located between staggered contralateral hemisections at the right T1/T2 and
left T10/T11 junctions. A1: haloperidol (40 ␮M) failed to block rhythmic
activity induced by electrical stimulation of the brain stem. Note that T8
ventral root rather than L5 recordings were used in this example and that T8
and L2 recordings from the same side are normally coactive. A2: a higher
concentration of haloperidol (80 ␮M) did block rhythmic activity. A3: after
haloperidol washout, lumbar rhythm activity reappeared, although rhythmic
activity in the thoracic segment (T8) remained suppressed.
of neurochemical substances, alone or in combination (see
Introduction). In addition, intrathecal injection of serotonergic
and noradrenergic (␣1) agonists in the lumbar region has been
shown to improve the voluntary locomotor pattern in cats with
partial spinal cord injury at T13 (Brustein and Rossignol 1999).
Unique to the present study, locomotor circuitry in the
hindlimb enlargement was excluded from exposure to bathapplied agonists. Excitatory agents were applied exclusively to
the thoracic region, which presumably contains propriospinal
neurons involved in descending transmission of the locomotor
command signal to the lumbar region.
In the second part of the study, using neurochemical excitatory agents, monoaminergic receptor stimulation facilitated
locomotor signal propagation, consistent with the ability of
corresponding serotonergic, dopaminergic, and noradrenergic
J Neurophysiol • VOL
Neurochemical
Agent or
Manipulation
Concentration Range,
␮M unless otherwise
stated
Brain Stem-Evoked
Rhythmic Activity
Facilitated
5-HT ⫹ NMDA
NMDA
Mg2⫹ removal
5-HT
Quipazine
8-OH-DPAT
NE
Methoxamine
Clonidine
Dopamine
Dopamine ⫹ NE
ACh ⫹ edrophonium
[K⫹] increase
(10–50) ⫹ (2–5)
2–4
0
10–50
10–20
1–30
15–60
40–100
30–60
100–500
400 ⫹ 50
(20–50) ⫹ 100
Target: 7–9 mM
3/9
0/14
0/9
13/30
3/7
2/10
1/28
5/11
0/5
1/15
6/9
0/11
2/7
The capacity of each agent or manipulation to facilitate brain stem-evoked
rhythmic activity is indicated as the number of preparations out of the total in
which locomotor-like activity developed. All long direct bulbospinal projections were disrupted by staggered contralateral hemisections at the right T1/T2
and left T10/T11 junctions. All of these preparations were selected on the basis
of failure to produce locomotor-like activity in response to brain stem stimulation alone. 5-HT, 5-hydroxytryptamine; NMDA, N-methyl-D-aspartate;
8-OH-DPAT, 8-hydroxy-2-(di-n-propylamino)tetralin; NE, norepinephrine;
ACh, acetylcholine; [K⫹], K⫹ concentration.
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antagonists to suppress such transmission. The fact the muscarinic receptor activation failed to enhance signal transmission is congruent with muscarinic receptor blockade having no
effect on locomotor-like discharge in preparations responsive
to brain stem stimulation alone. However, attempts to enhance
NMDA receptor-mediated actions, either by application of
NMDA or by increasing channel conductance (removal of
Mg2⫹ ions), failed to facilitate bulbospinal transmission, despite
the fact that NMDA receptor blockade in the thoracic region
suppressed brain stem-induced rhythmic activity. This disparity
may be due to interference by nonspecific widespread neuronal
excitation, given the ubiquitous distribution of NMDA receptors
among spinal neurons. Therefore, it seems important to establish
neuronal excitability at an appropriate level to facilitate locomotor
command signal transmission. In the present series this was not
successfully accomplished using NMDA application or Mg2⫹ ion
removal. Similarly, in an earlier study involving neurochemical
manipulation of the whole cord, Mg2⫹ ion removal failed to
promote locomotor-like activity, an observation thought to be due
to excessive activation of NMDA receptors (Cowley et al. 2005).
One limitation of the approach used in this study to facilitate
bulbospinal transmission is the time course of receptor stimulation. Application of neurochemicals to the in vitro bath
produces tonic long-lasting stimulation, whereas during natural
behavior, endogenous release of neurochemicals has a physiological temporal profile that is no doubt quite different.
Another limitation is that in addition to eliminating direct
long-projecting bulbospinal pathways, staggered bilateral hemisections also interrupt, at least partially, the propriospinal
system itself. Both of these factors may contribute to the
observation that among agents showing a positive facilitatory
effect on locomotor rhythm generation, facilitation did not
occur in all preparations exposed to such agents.
FACILITATION OF LOCOMOTOR COMMAND SIGNAL TRANSMISSION
J Neurophysiol • VOL
Fig. 7. Brain stem-evoked rhythmic activity was facilitated by stimulation of
noradrenergic but not N-methyl-D-aspartate (NMDA) receptors located on
thoracic propriospinal neurons. A1: electrical stimulation of the brain stem
alone failed to elicit rhythmic activity. A2: application of NMDA to thoracic
cord segments (T3–T9) located between staggered contralateral hemisections
(right T1/T2 and left T10/T11) failed to facilitate any clear pattern of rhythmic
activity in response to brain stem stimulation. A3: the ␣1-noradrenergic
receptor agonist methoxamine promoted brain stem-evoked rhythmic activity
in thoracic and lumbar segments. Ipsilateral thoracic T8 and L2 ventral roots
are usually coactive (see Fig 5).
Selective neurochemical stimulation of cervical segments
produces locomotor-like activity in both the cervical and lumbar regions of the in vitro neonatal rat preparation (Ballion et
al. 2001; Cowley and Schmidt 1997; Cowley et al. 2008; Juvin
et al. 2005) as well as cervical segments of the mudpuppy
(Wheatley and Stein 1992). Axial muscles supplied by thoracic
segments are also rhythmically active during locomotion in rats
(Gramsbergen et al. 1999), cats (Carlson et al. 1979; Koehler et
al. 1984; Zomlefer et al. 1984), and humans (de Seze et al.
2008; Thorstensson et al. 1982). Earlier studies proposed that
locomotor rhythm generators in the neonatal rat were restricted
to a limited number of spinal segments in the lumbar and
cervical regions and suggested that rhythmic output of thoracic
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Fig. 6. Facilitation of brain stem-evoked rhythmic activity in the lumbar region by
stimulation of 5-HT receptors located on thoracic propriospinal neurons. A1: in this
preparation, electrical stimulation of the brain stem alone failed to elicit locomotor-like
activity. A2: application of 5-HT to thoracic cord segments (T3–T9) located between
staggered contralateral hemisections (right T1/T2 and left T10/T11) enabled locomotor-like activity to appear in response to brain stem stimulation. A3: the facilitatory
effect of 5-HT was abolished after washout. B: the ability to induce locomotor-like
activity depended on both the brain stem stimulus intensity and concentration of 5-HT
applied to the thoracic cord. Three different concentrations of 5-HT (10, 30, and 50
␮M) were applied at 4 different levels of brain stem stimulus (1, 2, 3, and 4 mA) in 7
preparations. At 1 mA, only the high concentration of 5-HT (50 ␮M) was effective
(n ⫽ 1/7). At high stimulation intensity (4 mA), all three 5-HT concentrations (10, 30,
and 50 ␮M) enabled locomotor-like activity in preparations unresponsive to brain stem
stimulation alone; however, higher 5-HT concentrations were effective in more
preparations (n ⫽ 3/7, 4/7, and 5/7 for 10, 30, and 50 ␮M, respectively). The y-axis
denotes the number of preparations displaying locomotor-like activity among the 7
preparations tested at each concentration and stimulus strength.
2825
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FACILITATION OF LOCOMOTOR COMMAND SIGNAL TRANSMISSION
segments is passively driven by circuitry in the cervical and
lumbar regions (Ballion et al. 2001; Cazalets et al. 1995). In
contrast, we previously provided data suggesting that a 5-HTsensitive oscillatory network, capable of producing a locomotor output, was distributed in the spinal cord, including the
thoracic and cervical regions (Cowley and Schmidt 1997).
Other studies support the concept of locomotor-related rhythmogenic elements distributed throughout the spinal cord in
vertebrates ranging from lamprey to humans (Ceccato et al.
2009; Falgairolle et al. 2006; Grillner 1981; Hagevik and
McClellan 1999). Within such a longitudinally distributed
system, gradients of enhanced rhythmogenesis that are centered in the forelimb and hindlimb enlargements are suggested
by the available data (e.g., Ballion et al. 2001; Cazalets et al.
1995; Cowley and Schmidt 1997; Kjaerulff and Kiehn 1996;
Kremer and Lev-Tov 1997). Assuming that oscillatory components are present throughout the spinal cord and that local
circuits need to communicate with each other, the rhythmgenerating elements of a distributed locomotor network and the
propriospinal neurons transmitting the descending command
signal may be one and the same, or at least substantially
overlap. Thus, during brain stem stimulation, neurochemicals
applied to the thoracic region may promote hindlimb stepping
by enhancing propriospinal transmission of a tonic descending
brain stem command signal or by recruiting activity in the
thoracic portion of a widely distributed rhythmic network, or a
combination of both mechanisms.
As highlighted by Rossignol et al. (2001), the effect of drug
administration on locomotion in experimental animals depends
J Neurophysiol • VOL
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Fig. 8. Facilitation of brain stem-evoked rhythmic activity in the lumbar region
by stimulation of dopaminergic and noradrenergic receptors located on thoracic propriospinal neurons. A1: application of dopamine and norepinephrine
(NE) to thoracic cord segments (T3–T9) located between staggered contralateral hemisections (right T1/T2 and left T10/T11) promoted locomotor-like
activity in response to brain stem stimulation. A2: electrical stimulation of the
brain stem in the absence of dopamine and norepinephrine failed to elicit
rhythmic activity in the same preparation.
on a variety of factors, not the least of which is the type of
preparation. For example, the ␣2-noradrenergic receptor agonist clonidine promotes locomotion in the complete spinal cat
(e.g., Forssberg and Grillner 1973; Marcoux and Rossignol
2000), has a deleterious effect in partial spinal cats (Brustein
and Rossignol 1999; Giroux et al. 1998), and fails to elicit
locomotion in the in vitro neonatal rat spinal cord (SqalliHoussaini and Cazalets 2000) or in mice with complete thoracic cord transection (Lapointe et al. 2008). Consistent with
the previous reports using rodent preparations, clonidine failed
to facilitate propriospinal transmission of the locomotor signal
when applied to the thoracic region during brain stem stimulation. However, the ␣2-noradrenergic receptor antagonist yohimbine blocked locomotor-like activity in preparations capable of responding to brain stem stimulation alone. These
incongruent observations, using ␣2-noradrenergic receptor
agonists and antagonists, resemble the disparate results for
NMDA receptor agonists vs. antagonists noted above. In the
case of yohimbine, 5-HT1A receptor-mediated inhibitory actions, in addition to ␣2-noradrenergic receptor blockade, might
be considered. The proposal that yohimbine (when applied
from C5 to the conus) suppresses brain stem-evoked locomotor-like activity through activation of 5-HT1A receptors, which
in turn exerts an inhibitory modulation of locomotor rhythm
frequency (Beato and Nistri 1998), was suggested by Lui and
Jordan (2005). They invoked this 5-HT receptor-dependent
mechanism to help explain why, unlike yohimbine, the more
specific ␣2-noradrenergic receptor antagonist RX 82002 failed
to block locomotion. However, 5-HT1A receptor activation
alone does not adequately account for the effect of yohimbine
observed in our experiments; in particular, the 5-HT1 receptor
agonist 8-OH-DPAT facilitated propriospinal transmission in 2
of 10 preparations.
Complex actions of different noradrenergic receptor subtypes at pre- and postsynaptic levels may also contribute to the
incongruent results regarding the ␣2-noradrenergic receptor
agonist (clonidine) vs. antagonist (yohimbine). Agonists of ␣2and ␤-noradrenergic receptors are known to presynaptically
inhibit glutamatergic inputs to lumbar motoneurons in the
neonatal rat (Tartas et al. 2010) and decrease rhythm frequency
(Kiehn et al. 1999; Sqalli-Housaini and Cazalets 2000). In
contrast, agonists of all three major noradrenergic receptor
subtypes (␣1, ␣2, and ␤) increase motoneuronal membrane
excitability (Tartas et al. 2010). It is unclear whether these
findings in lumbar motoneurons apply to other neurons such as
thoracic propriospinal cells. Possibly clonidine does not increase thoracic propriospinal neuron excitability, or if increased excitability occurs, it is insufficient to facilitate locomotor signal propagation and/or is counterbalanced by presynaptic inhibition (Tartas et al. 2010). Similarly, norepinephrine
application may be generally ineffective because of a dominance of presynaptic inhibitory actions (via ␣2- and ␤-noradrenergic receptors). On the other hand, the ␣1-agonist methoxamine, unlike ␣2- and ␤-agonists, potentiates presynaptic excitatory glutamatergic drive, in addition to increasing membrane
excitability (Tartas et al. 2010). Thus the combination of ␣1receptor-mediated pre- and postsynaptic excitatory effects may
explain why methoxamine facilitated locomotor-like activity in
45% of the preparations tested, whereas norepinephrine itself,
which activates all three receptors, was rarely (1/28 preparations)
effective.
FACILITATION OF LOCOMOTOR COMMAND SIGNAL TRANSMISSION
J Neurophysiol • VOL
responsiveness to neurochemicals is anticipated in the chronic
state because of compensatory changes in the nervous system
(Rossignol 2006; Rossignol et al. 2009). Other investigators
have started to explore complementary methods of facilitating
locomotor-related propriospinal activity. These include the use
of neurotrophic factors to strengthen synaptic connections in
the staggered contralateral hemisected neonatal rat (Arvanian
et al. 2006a, 2006b) and tonic electrical stimulation of propriospinal neurons caudal to complete chronic T8/T9 transection in
the rat (Yakovenko et al. 2007). A combined approach may
well offer the best chance of improving function for patients
with spinal cord injury, as discussed in recent literature (e.g.,
Gerasimenko et al. 2007; Musienka et al. 2009).
GRANTS
This work was supported by the Canadian Institutes of Health Research.
K. C. Cowley was supported by the Will-to-Win Scholar Fund.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
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Why the combination of dopamine and norepinephrine was
considerably more effective in facilitating propriospinal transmission of the locomotor command signal (6/9 preparations)
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