1Section for Neurophysiology, Department of Physiology, The Panum Institute, 2200 Copenhagen N, Denmark; and 2Gatty Marine Laboratory, School of Biomedical Sciences, University of St. Andrews, St. Andrews, Fife KY103SS, Scotland
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ABSTRACT |
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Kiehn, Ole, Keith T. Sillar, Ole Kjaerulff, and Jonathan R. McDearmid. Effects of Noradrenaline on Locomotor Rhythm-Generating Networks in the Isolated Neonatal Rat Spinal Cord. J. Neurophysiol. 82: 741-746, 1999. We have studied the effects of the biogenic amine noradrenaline (NA) on motor activity in the isolated neonatal rat spinal cord. The motor output was recorded with suction electrodes from the lumbar ventral roots. When applied on its own, NA (0.5-50 µM) elicited either no measurable root activity, or activity of a highly variable nature. When present, the NA-induced activity consisted of either low levels of unpatterned tonic discharges, or an often irregular, slow rhythm that displayed a high degree of synchrony between antagonistic motor pools. Finally, in a few cases, NA induced a slow locomotor-like rhythm, in which activity alternated between the left and right sides, and between rostral and caudal roots on the same side. As shown previously, stable locomotor activity could be induced by bath application of N-methyl-D-aspartate (NMDA; 4-8.5 µM) and/or serotonin (5-HT; 4-20 µM). NA modulated this activity by decreasing the cycle frequency and increasing the ventral root burst duration. These effects were dose dependent in the concentration range 1-5 µM. In contrast, at no concentration tested did NA have consistent effects on burst amplitudes or on the background activity of the ongoing rhythm. Moreover, NA did not obviously affect the left/right and rostrocaudal alternation of the NMDA/5-HT rhythm. The NMDA/5-HT locomotor rhythm sometimes displayed a time-dependent breakdown in coordination, ultimately resulting in tonic ventral root activity. However, the addition of NA to the NMDA/5-HT saline could reinstate a well-coordinated locomotor rhythm. We conclude that exogenously applied NA can elicit tonic activity or can trigger a slow, irregular and often synchronous motor pattern. When NA is applied during ongoing locomotor activity, the amine has a distinct slowing effect on the rhythm while preserving the normal coordination between flexors and extensors. The ability of NA to "rescue" rhythmic locomotor activity after its time-dependent deterioration suggests that the amine may be important in the maintenance of rhythmic motor activity.
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INTRODUCTION |
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The neural networks of the vertebrate spinal cord
that are responsible for generating rhythmic locomotor movements are
capable of producing a wide spectrum of outputs in which the intensity, duration, cycle frequency, and phasing of the motor bursts are variable
(Kiehn et al. 1997; Sillar et al. 1997
).
This flexibility in motor output is important because rhythmic
movements (swimming or walking, for example) must be able to adapt to
changing behavioral requirements. Some of this flexibility in motor
rhythm generation is accomplished by inputs from peripheral sense
organs. However, the motor output can also be changed centrally through
neuromodulation of the spinal networks themselves. Within the rhythm
and pattern-generating networks, neuromodulators can act on both the
electrical properties of neurons and on their synaptic interconnections
(Kiehn and Katz 1999
). Some of the neuromodulators are
intrinsic to the spinal networks themselves, whereas others originate
in centers outside the spinal cord such as the brain stem (Katz
1995
). Prominent among the extrinsic sources of neuromodulation
are noradrenergic (NA) and serotonergic (5-HT) neurons of locus
coeruleus and the raphe nuclei, respectively.
There is now a wealth of information on the actions of 5-HT on spinal
motor networks. 5-HT is able to initiate (Cazalets et al.
1992; Kiehn and Kjaerulff 1996
; Viala and
Buser 1969
), or modulate (Barbeau and Rossignol
1991
; Harris-Warrick and Cohen 1985
;
Sillar et al. 1992
) locomotor activity in all
vertebrates studied so far. These actions, mediated at a range of 5-HT
receptor subtypes (Wallis 1994
), are accomplished via
effects of 5-HT on cellular properties (Sillar and Simmers
1994
; Wallén et al. 1989
) and synaptic
strengths (McDearmid et al. 1997
). Although less is
known in detail about the actions of NA on spinal networks, this amine
is also able to initiate stable locomotor activity in the cat and
rabbit (Barbeau and Rossignol 1991
; Chau et al. 1998
; Forssberg and Grillner 1973
;
Jankowska et al. 1967
; Kiehn et al. 1992
;
Viala and Buser 1969
) and to slow down the ongoing locomotor rhythm in the cat (Rossignol et al. 1998
) and
the Xenopus tadpole (McDearmid et al.
1997
). In the tadpole, a relatively simple lower vertebrate
preparation, NA strengthens reciprocal inhibition via a presynaptic
facilitation of glycine release from commissural interneurons
(McDearmid et al. 1997
). It has been proposed that this
mechanism is largely responsible for the slowing effect of NA on
swimming. However, whether this modulatory action on glycinergic
synapses is phylogenetically conserved in higher vertebrates, or
whether other cellular mechanisms may be involved in the slowing effect
of NA on locomotor activity is still unknown. The purpose of the
present study, therefore was to initiate a study on the cellular,
synaptic, and network actions of NA in the spinal cord of higher
vertebrates. For this purpose we have used a relatively simple
mammalian preparation, the isolated spinal cord of the neonatal rat.
The effects of NA (unlike 5-HT) on locomotor activity in the neonatal
rat have yet to be addressed in any detail (Cazalets et al.
1990
). We therefore have begun to examine the modulatory role
of NA on rhythm and pattern generation in the neonatal rat, by
investigating the transmitter's ability to induce rhythmic motor
activity and to modulate various parameters of the ongoing locomotor pattern.
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METHODS |
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Dissection
Preparations of the spinal cord (n = 14) were
isolated from neonatal rats 0-2 days old. Details of the dissecting
procedure have been described previously (Kiehn and Kjaerulff
1996), but, briefly, the rats were first deeply anesthetized
with ether, then decapitated and eviscerated. The spinal cord extending
from C1 to L6, including
ventral and dorsal roots, was then isolated and the preparation
transferred to a recording chamber and superfused with oxygenated (95%
O2-5% CO2) Ringer solution
was of the following composition (in mM): 128 NaCl, 4.7 KCl, 1.2 KH2PO2, 1.25 MgSO4, 25 NaHCO3, 2.5 CaCl2, and 20 glucose, pH 7.4. Experiments were performed at room temperature.
Induction of rhythmic locomotor activity
Rhythmic locomotor activity was induced in the spinal cord by
the bath application of N-methyl-D-aspartic acid
(NMDA, 4-8.5 µM) alone or in combination with 5-hydroxytryptamine
(5-HT, 4-20 µM). With the composition of the extracellular medium
used in our laboratory, these drug concentrations usually give stable long-lasting locomotor-like activity (see below and Iizuka et al. 1997; Kiehn and Kjaerulff 1996
;
Kjaerulff and Kiehn 1996
). The preparation would be
considered healthy as long as 5-HT/NMDA could produce a locomotor-like
motor output. Norepinephrine (NA, 0.5-50 µM) was added either on its
own (Fig. 2), together with NMDA/5-HT from the start of the experiment
(Fig. 3), or during ongoing activity induced by administration of 5-HT
and/or NMDA (Fig. 5). All drugs were obtained from Sigma (St. Louis, MO).
Recordings
Activity in the L2 and
L5 ventral roots on both sides of the cord was
recorded with suction electrodes. Activity in these roots corresponds
to flexor and extensor activity, respectively (Kiehn and
Kjaerulff 1996). Root recordings were band-pass filtered (100-10,000 Hz), digitized, stored on a digital tape recorder (Biologic DTR 1800; Claix, France), and printed on thermosensitive paper (Gould 4000; Valley View, OH).
Analysis
Ventral root recordings were digitized (1,000 Hz) off-line,
sampled by the Axoscope software program (version 7.0, Axon
Instruments), and analyzed using a custom-designed program written in
the Matlab (The MathWorks) environment. To characterize the rhythm
quantitatively we measured several parameters of the ventral root
activity under each experimental condition. The ventral root signal
(usually in L2, in which the amplitude modulation
is often most clear) was rectified and low-pass filtered. The minimal
voltage (Vval, for valley) and the corresponding time
(Tval) were determined in each locomotor cycle (Fig.
1C). The locomotor cycle
duration was defined as the time between the minimal voltage in
consecutive cycles (Tvaln+1 Tvaln, Fig. 1C). The peak
voltage (Vpk), and the time,
Thalfu, taken to reach the voltage
halfway between Vval and Vpk on the up-slope of
the burst, and the corresponding Thalfd on
the down-slope of the burst were also used.
Thalfd
Thalfu (the half-width) was used to
measure the ventral root burst duration. Vval and
Vpk
Vval defined the interburst amplitude and the burst amplitude modulation, respectively. Mean values in
individual experimental conditions were obtained by averaging between
10 and 40 consecutive cycles. All parameters were normalized by
dividing with control values, and statistical significance between
control and NA applications were tested with Student's t-test. Differences in the NA effects for different ranges
of drug concentrations were tested by ANOVA. The level of statistical significance was set at 0.05. Values in the text are means ± SD.
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RESULTS |
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NA-induced motor activity
NA was applied in 31 experiments at concentrations in the range of
0.5-50 µM (8.9 ± 12.0 µM, mean ± SD). NA elicited no
ventral root response (n = 8; 3.6 ± 6.7 µM;
range 0.5-20 µM; Fig. 2A), tonic ventral root activity (n = 5; 5.4 ± 8.2 µM; range 1-20 µM), or a rhythmic motor output (n = 18; 11.6 ± 13.7; range 1-50 µM; Fig. 2, B-C).
There was no significant ascending concentration dependency for these
responses in the population of animals examined. However, such a
dependency was found in individual animals (Fig. 2), where low
concentrations of NA elicited no ventral root activity, higher
concentrations evoked tonic discharge, and the highest concentrations
led to rhythmic activity. There was no obvious relationship between the
cycle period and the transmitter concentration. The rhythmic motor
responses were characterized by relatively long cycle periods of
42.4 ± 24.8 s (range 4-76 s, determined 8-10 min after
onset of transmitter superfusion). In two experiments the NA-induced
motor rhythms involved regular left-right (lL2-rL2) and rostrocaudal
(l-L2/l-L5 and r-L2/r-L5) alternation, similar to (but slower than;
cycle periods 4-14 s) the rhythmic activity observed with 5-HT/NMDA
(e.g., Fig. 1B) or 5-HT and NMDA alone (Cazalets et
al. 1992; Cowley and Schmidt 1994
;
Kjaerulff and Kiehn 1996
). In the remaining experiments
the rhythm was often irregular with a large propensity for synchronous
bursts in roots that usually alternate, but with alternation
intermingled. An example of this is shown in Fig. 2C where
the large bursts in l-L2/r-L2 are synchronous, as are the lower
amplitude bursts in all four roots. However, the large bursts are also
alternating with the smaller bursts. These experiments with NA alone
show that this transmitter is able to trigger a rhythmic motor output with a coordination that in most cases does not resemble any of the
previously described transmitter-induced rhythms in the neonatal rat
spinal cord (see Kiehn et al. 1997
).
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Noradrenergic modulation of an ongoing rhythm
NMDA and 5-HT were first applied to induce long-lasting rhythmic
activity so that modulatory effects of bath-applied NA on locomotor
activity could be investigated. In the experiments included in this
study the rhythmic activity was always alternating between L2 ventral roots on either side of the cord and
between L2 and L5 on the
same side of the cord. We will refer to this coordination pattern as
locomotor activity (Cowley and Schmidt 1994;
Iizuka et al. 1997
; Kiehn and Kjaerulff
1996
). As has been reported previously, the 5-HT/NMDA-induced
locomotor activity recorded in the neonatal rat shows time-dependent
changes in frequency (Sqalli-Houssaini et al. 1993
).
Usually the cycle periods are long initially and then progressively
shorten until they reach a plateau ~4-6 min after the initial bursts
are observed. The sequences of locomotor activity used for analysis in
the present study were taken during this plateau phase at comparable
times after the onset of the transmitter perfusion (5-HT/NMDA alone or
in combination with NA). There was always a period of at least 15 min
wash in control saline between drug applications. During the wash out
period the ventral root activity would vanish and eventually disappear
usually 5-10 min after initiating the wash. In some experiments NA was added during the plateau phase of a 5-HT/NMDA- or NMDA-induced rhythm.
Sequences of locomotor activity taken from this period were then
compared with equivalent locomotor sequences when a new plateau phase
was reached. Under these conditions NA significantly (Student's
t-test) increased cycle periods compared with control values
(n = 22, Figs. 3 and
4). The strength of this slowing effect increased in the range 1-5 µM (control cycle periods normalized to
100%; 1 µM, n = 7, cycle period = 136 ± 34%; 2-5 µM, n = 6, 169 ± 52; Figs. 3,
A-C, and 4A; the population data for 1 µM and 2-5 µM were not significantly different, ANOVA) but with an apparent ceiling at higher concentrations (>5 µM, n = 9, 164 ± 47, Fig. 4A). The slowing effect could be
detected at NA concentrations, which produced no motor responses when
applied alone (Fig. 3E). The effect was reversible (wash,
n = 10, 95 ± 16, Figs. 3D and 4A). In general the changes in period were accompanied by a
concentration-dependent increase in burst duration (1 µM, 134 ± 44; 2-5 µM, 164 ± 52; >5 µM 154 ± 54; wash, 97 ± 20, Figs. 3, A-C, and 4B). However, in the
presence of NA the duty cycle (burst duration divided by period length)
was comparable with that of control so that burst durations and cycle
periods seem to co-vary. The other locomotor parameters, that is the
burst amplitude (Vpk-Vval; Fig. 4C) and the
amplitude of the interburst activity (Vval; not illustrated in Fig. 4), did not change systematically, although there was a
tendency for the burst amplitude to increase in some but not all
ventral roots at higher NA concentrations (see Fig. 3,
A-C). Finally, the left/right and rostrocaudal alternation
of the NMDA/5-HT rhythm was not obviously affected by NA application at
any concentration tested (Fig. 3, A-C). We conclude from
these experiments that NA has a slowing effect on the ongoing rhythm
but has little effect on other locomotor parameters.
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Restoration of rhythmic locomotor activity by NA
In the majority of experiments, the cycle periods of the NMDA/5-HT
or NMDA rhythms started out with initially long periods and then
shorten until they reach a plateau (see previous section). In some
experiments the progressive shortening of the cycle periods of
locomotor episodes induced by these drugs (Fig.
5A and B)
was followed by a complete breakdown of the rhythm (Fig.
5C), which deteriorated into tonic activity (see also
Sqalli-Houssaini et al. 1993). However, bath
applications of NA (still in the presence of NMDA/5-HT; Fig.
5D) were able to restore a well-coordinated locomotor
rhythm, similar to that occurring before the breakdown but usually at a
lower average frequency. The changes in cycle duration (Fig.
5E,
) for this episode of events are shown in Fig.
5E along with the changes in burst duration (Fig.
5E,
). Corresponding decreases or increases in burst
duration followed the variations in cycle duration. Figure
5F shows the changes in peak burst amplitude
(Vpk,
), and interburst amplitude (Vval,
) for the same episode. Note that the peak amplitude
decreases while the interburst amplitude increases before the
breakdown, leading to a decrease in modulation amplitude. NA restored
the modulation amplitude.
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This "rescuing" of the locomotor activity was seen in five of seven experiments for NA concentrations in the range 1-20 µM (7.0 ± 6.3 µM). In most cases the restoration of the rhythm by NA lasted for the duration of the NA application (10-15 min), although in one case it only lasted for ~3-4 min, after which the rhythm broke down again.
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DISCUSSION |
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In this study we have shown that in the neonatal rat spinal cord, NA can trigger either tonic activity or a rhythmic motor pattern that is dominated by coactivation in ventral roots. NA also has a robust slowing effect on ongoing locomotor rhythms even at concentrations that are insufficient on their own to trigger any motor output. In addition, the NMDA/5-HT-induced rhythms can deteriorate into tonic activity after prolonged drug exposure, yet a robust and slower rhythm would resume after introduction of NA. Together these observations suggest that NA may be an important modulator of mammalian locomotor activity.
NA activation and modulation of locomotor activity
The rhythmic motor pattern evoked by higher concentrations of NA
in the neonatal rat involves coactivation of functionally antagonistic
motor roots and thus differs from the more regular alternating
locomotor activity seen in adult spinal mammals after injection of the
catecholamine precursor L-dihydroxyphenylalanine (L-DOPA) (Grillner and Zangger 1979;
Jankowska et al. 1967
; Viala and Buser
1969
) or clonidine (Barbeau and Rossignal 1991
;
Forssberg and Gillner 1973
) or after intrathecal
application of NA itself or
-receptor agonists (Chau et al.
1998
; Kiehn et al. 1992
). It also differs from
the previously described alternating and much faster rhythmic motor
patterns in the neonatal rat induced by 5-HT, glutamate-receptor
agonists, and dopamine (Cazalets et al. 1990
,
1992
; Cowley and Schmidt 1994
;
Kiehn and Kjaerulff 1996
; Kudo and Yamada
1987
). However, the NA-induced rhythm bears some resemblance to
the synchronous activity evoked by acetylcholine (Cowley and
Schmidt 1994
) and by strychnine in combination with bicuculline
(Bracci et al. 1996
; Cowley and Schmidt
1995
).
Because the high degree of synchrony together with the relatively long
cycle periods of the NA-induced rhythm are not well suited to perform
normal locomotion, it seems more likely that the main role of NA in the
neonatal rat is to modulate ongoing motor activity. In this respect the
slowing of the locomotor rhythm by NA, accompanied by an increase in
burst durations, but with little effect on other locomotor parameters,
is similar to the effects previously described in the cat
(Rossignol et al. 1998). Interestingly, the slowing
effect occurred without obvious changes in the coordination of the
ongoing 5-HT/NMDA-induced locomotor activity, despite the fact that NA
alone could produce a slow synchronous motor pattern very distinct from
the 5-HT/NMDA rhythm. Similarly, when NA rescued the 5-HT/NMDA rhythm
following its time-dependent rundown, this rhythm always reverted to a
pattern similar in its coordination to the one present before the
rundown. Furthermore, a characteristic of this modulatory effect of NA is that the rescued rhythm was always slower than the 5-HT/NMDA rhythm
observed before deterioration. So it appears that when there is an
ongoing rhythm in the presence of 5-HT/NMDA, the initiating role of NA
is disguised by its modulatory slowing role.
The observation that NA can rescue rhythmic activity after prolonged exposure to 5-HT and NMDA may be useful for future experimental studies on the neonatal rat because it allows for longer-lasting stable locomotor activity. More importantly, this observation also raises the possibility that endogenously released NA, possibly originating from neurons in the locus coeruleus, could play a role in sustaining locomotor rhythm generation in the intact animal.
Mechanisms for the NA modulation
In the Xenopus tadpole, NA causes a slowing of the
cycle period during swimming, with little effect on the burst durations (McDearmid et al. 1997) but a pronounced effect on the
longitudinal coordination of the motor output (J. McDearmid, O. Kjaerulff, O. Kiehn, C. A. Reith, and K. T. Sillar,
unpublished observations). At the cellular level, NA increases the
amplitude of reciprocal, mid-cycle inhibition by enhancing glycinergic
transmission in the network through a direct presynaptic facilitation
of the release mechanism (McDearmid et al. 1997
). It has
therefore been proposed that NA slows the swimming rhythm by prolonging
the inhibitory phase of the swimming cycle and so delaying the burst
onsets (McDearmid et al. 1997
). It is conceivable that a
similar enhancement of reciprocal glycinergic transmission, which is
known to contribute to spinal rhythm generation in the neonatal rat
(Cowley and Schmidt 1995
), could contribute to the
slowing effect of NA on the locomotor rhythm. However, two observations
suggest that additional effects of NA on the spinal networks are likely
to be involved. First, in the absence of 5-HT/NMDA, NA can trigger both
tonic and slow rhythmic activity, indicating an excitatory influence on
spinal cord neurons. Second, the NA-induced slowing of the 5-HT/NMDA rhythm is not accompanied by any change in the amplitude of interburst activity, as one would predict to result from enhanced reciprocal inhibition (cf. McDearmid et al. 1997
). These and other
effects of NA on rhythm-generating elements in the network await
exploration using intracellular recordings from motor and interneurons.
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ACKNOWLEDGMENTS |
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We thank M. Tresch for implementing the parameter analysis in Matlab and D. McLean for producing the drawings in Fig. 1A.
This work was enabled by a twinning grant from The European Neuroscience Program to O. Kiehn and K. T. Sillar. This work was also supported by a grant from the Novo foundation to O. Kiehn's laboratory, by a Wellcome Trust grant to K. T. Sillar, and by an earmarked studentship to J. R. McDearmid from the biotechnology and Biological Sciences Research Council.
Present addresses: O. Kjaerulff, Nobel Institute for Neurophysiology, Karolinska Institutet, Doktorsringen 12, 171 77 Stockholm, Sweden; J. R. McDearmid, Dept. of Physiology and Biophysics, Mount Sinai Medical School, One Gustav L. Levy Place, Box 1218, New York, NY 10029.
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FOOTNOTES |
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Address for reprint requests: O. Kiehn, Section of Neurophysiology, Dept. of Physiology, The Panum Institute, Blegdamsvej 3, 2200 Copenhagen N, Denmark.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 25 March 1999; accepted in final form 5 May 1999.
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REFERENCES |
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