Biophysics Sector and Istituto Nazionale di Fisica della Materia Unit, International School for Advanced Studies (SISSA), 34014 Trieste, Italy
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ABSTRACT |
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Beato, M. and A. Nistri. Interaction Between Disinhibited Bursting and Fictive Locomotor Patterns in the Rat Isolated Spinal Cord. J. Neurophysiol. 82: 2029-2038, 1999. Using a transverse barrier that allowed discrete application of neurochemicals to certain lumbar regions of the rat isolated spinal cord, we studied the intersegmental organization of rhythmic patterns recorded extracellularly from ventral roots and intracellularly from single motoneurons. Fictive locomotor patterns were elicited by serotonin (5-HT) and/or N-methyl-D-aspartate (NMDA) or high K+ solution applied to the rostral or caudal lumbar region of the cord. Neither 4-aminopyridine nor Mg2+-free solution shared this property. Coapplication of strychnine and bicuculline (blockers of fast synaptic inhibition) to the caudal part elicited slow bursting in the whole cord. These bursts could trigger episodes of fictive locomotion patterns in the rostral roots. When the rostral region was exposed to 5-HT and/or NMDA (during continuous application of strychnine and bicuculline caudally) a standard locomotor-like pattern was generated during each interburst interval and was surprisingly expressed with its typical pattern alternation even in the caudal area despite the local presence of strychnine and bicuculline. Midsagittal splitting of the caudal region did not change this alternating pattern, indicating that it was driven by rostral regions above the surgical cut. Discrete changes in the concentrations of NMDA rostrally modulated the burst amplitude recorded in the same region after caudal application of strychnine and bicuculline. The period of fictive locomotor patterns changed bimodally depending on the temporal relation with disinhibited bursts, indicating a tight interaction between these two rhythmic activities. These results are interpreted on the basis of a model that assumes a modular arrangement for the locomotor central pattern generator, made up by a series of unit burst generators with serial and crossed connections.
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INTRODUCTION |
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In the neonatal rat spinal cord, in vitro
locomotor-like rhythmic activity (usually termed as "fictive
locomotion") is generated by a ventrally located premotoneuronal
network (Kjaerulff and Kiehn 1996) called central
pattern generator (CPG) and typically activated by agents such as
serotonin (5-HT) (Cazalets et al. 1992
),
N-methyl-D-aspartate (NMDA) (Kudo and
Yamada 1987
), or high K+ solutions
(Bracci et al. 1998
). The CPG is thought to be a modular assembly of functional units (unit burst generators) individually endowed with oscillatory activity transmitted to different muscles (Cheng et al. 1998
; Edgerton et al. 1976
;
Grillner 1981
; Grillner et al. 1991
). In
this neuronal network the coupling among unit burst generators would
then change in accordance with preprogrammed patterns to be expressed
as various stereotypic motor activities (Grillner 1985
;
Mortin and Stein 1989
; Svoboda and Fetcho
1996
). A major question is thus the nature of the CPG
mechanisms that allow switching a certain motor pattern on. Different
excitatory agents cannot apparently induce identical motor patterns as
shown by Kiehn and Kjaerulff (1996)
, who reported that
for instance the ventral root (VR) rhythmic activity induced by
dopamine closely resembles the coordinated muscle contractions observed
during locomotion, whereas the one elicited by 5-HT is closer to the muscle activity during swimming. In general, all these patterns are
characterized by left-right and flexor-extensor alternating oscillations (at 1- to 5-s period, recorded intracellularly from single
motoneurons or extracellularly from VR), although various muscle groups
are differentially involved according to distinct motor behaviors.
Block of synaptic inhibition does not prevent rhythmicity because
application of selective antagonists of GABAA
(Kremer and Lev-Tov 1997) or glycine receptors
(Cowley and Schmidt 1995
) can synchronize the
alternating oscillations detected in antagonist motor pools. Indeed,
full suppression of fast synaptic inhibition per se induces slow,
synchronous bursting (~30-s period) (Bracci et al.
1996a
,b
), which can be accelerated to the range of standard locomotion periods by the same agents used to induce locomotion (Bracci et al. 1996a
,b
). Furthermore, disinhibited
bursting shares with locomotor patterns the same sensitivity to block
of either class of ionotropic glutamate receptors (Beato et al.
1997
; Bracci et al. 1996a
,b
) and the same
anatomic location (Bracci et al. 1996b
; Kjaerulff
and Kiehn 1996
). These common features raise the possibility
that disinhibited rhythmic bursting might be generated by the same
oscillators that produce locomotor-like activity when their inhibitory
connections are intact. One alternative possibility is that distinct
networks are responsible for fictive locomotion and disinhibited rhythmicity.
The aim of the present experiments was to test whether a certain
rhythmic pattern generated at a given segmental level may influence the
operation of the CPG in adjacent spinal segments, if this interaction
is bidirectional and depends on intact inhibitory circuits. As a tool
to investigate these issues, the rat spinal cord in vitro was
partitioned with a vertical barrier lowered onto the cord at lumbar
level (Cazalets et al. 1995). Such a method allowed us
to study the interaction between distinct regions of the spinal cord in
which different rhythmic behaviors were pharmacologically induced. For
this purpose we simultaneously blocked synaptic inhibition in the
caudal spinal cord and applied locomotor substances to the rostral portion.
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METHODS |
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Standard procedures were used to obtain spinal cord preparations
of neonatal Wistar rats (0-4 days old) (Ballerini et al. 1997; Bracci et al. 1996a
). The isolated spinal
cord was fixed to the bottom of a recording chamber (~5 ml volume)
and continuously superfused (~5 ml/min) with artificial cerebrospinal
fluid (ACSF). The composition of the ACSF for both dissection and
recording was (in mM) 113 NaCl, 4.5 KCl, 1 MgCl27H2O, 2 CaCl2, 1 NaH2PO4, 25 NaHCO3, and 11 glucose, gassed with 95%
O2-5% CO2, pH 7.4. All
agents were superfused at the concentrations mentioned in the text.
The recording chamber contained a transverse barrier that enabled us to superfuse adjacent spinal regions separately via inlets at the rostral and caudal end of the bath. Excess solution outflowed via two independent drains. The barrier was made up by two asymmetric components always placed at the same segmental position, namely the junction between L4 and L3 segments. The spinal cord was first pinned with its dorsal side up to which a thin (<1 mm width, >10 mm long) hard plastic strip was fixed using special tissue glue (Embucrilate, Braun Chemicals, Melsungen, Germany) and then rotated ventral side up. A thin layer of petroleum jelly (Vaseline) was placed at the midline of the recording chamber across its whole width, and the plastic strip was then gently advanced through the Vaseline, which sealed it to the bottom of the recording chamber. A solid silicon barrier with an inverse U-shaped recess was subsequently lowered onto the cord in correspondence of the L3-L4 junction to complete the functional separation of the tissue in rostral and caudal portions. The thickness of the barrier was always less then one-third of the length of a segment. All leaks across the barrier were finally sealed up with Vaseline. The water tightness of the barrier was then tested by adding Phenol Red to one compartment and visually checking for any leakage of fluid from one compartment to the other. As a further precaution before starting the experiments, ACSF in one compartment was completely removed; the barrier was considered effective only if no fluid leak could be detected in this condition for at least 1 min. Leak tests were repeatedly performed during each experiment: whenever a leak was apparent, the experiment was interrupted. Caudal midsagittal lesions were performed on a number of preparations after having carried out control tests with the split bath configuration. In this case, test substances were flushed out and replaced with control ACSF while the lower part of the spinal cord was bisected with a miniature blade. After checking that the barrier had remained leak-proof, test substances were applied again.
DC-coupled extracellular recordings were simultaneously performed from
four VRs, namely pairs of L2 and
L5 VRs, with tight fitting suction pipettes
containing an Ag/Ag-Cl pellet. Intracellular recordings were obtained
from L2 motoneurons functionally identified by
stimulation of the corresponding VR. Sharp electrodes (40-60 M)
were filled with 3 M KCl, and recordings were performed in current-clamp configuration. During intracellular experiments VR
activity was usually monitored from three VRs only. All electrodes were
mounted on Narishige micromanipulators. Signals via extracellular electrodes were fed to a custom made four channels AC/DC amplifier (1,000 gain), monitored on a Gould chart recorder, and digitized and
recorded on a DAT tape for further off-line analysis.
Period and relative phase measurements were performed over at least 25 randomly chosen cycles to minimize short-term sampling bias. Period was
defined as the time between the onset of two cycles of locomotor
activity, whereas phase between two roots was defined as the latency
for the onset of a cycle in one root during the cycle of the other
root, divided by the period and expressed in angular degrees whereby
180° represent complete phase alternation and 0 or 360° full phase
coincidence (Kjaerulff and Kiehn 1996). All data were
quantified as means ± SD; statistical significance was assessed
with Student's t-test (unless otherwise stated) or ANOVA.
Forty-two spinal cord preparations were used for the present study. All
drugs were purchased from Sigma.
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RESULTS |
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Fictive locomotion induced by 5-HT, NMDA, or high potassium applied either at upper or lower lumbar level
As previously reported by Cazalets et al. (1995,
1996
), selective application of 5-HT and NMDA to rostral
segments elicited a locomotor pattern that could be detected from
rostral as well as caudal VRs. Figure 1
(top left) shows simultaneous recordings from four VRs in
the presence of 10 µM 5-HT and 2 µM NMDA when drugs were bath
applied to the rostral compartment only (segments from
T1 to L3). Within the first
2 min of application, we observed an upward baseline shift and a
pronounced thickening (not shown) in the recordings of
L2 VRs, which predominantly supply hindlimb flexor muscles (Kiehn and Kjaerulff 1996
). Such an
effect was undetectable in recordings from L5 VRs
[mainly supplying hindlimb extensor muscles (Kiehn and
Kjaerulff 1996
)], thus further confirming that drugs could not
diffuse across compartments. After 4 min superfusion with 5-HT and
NMDA, a rhythmic alternating pattern appeared in
L2 and L5 pairs, with
typical phase-lock of
lL5-rL2 and
rL5-lL2 (Fig. 1, top
left; period 3.5 ± 0.9 s). In 42/42 preparations in
which the barrier was placed at the
L3-L4 junction,
oscillatory activity was detected in both rostral and caudal segments
when the rostral cord only was exposed to various concentrations of NMDA (2-8 µM), 5-HT (5-30 µM), or a combination of these two
agents. Because activation of the rostral segments can induce an
alternating pattern in the more caudal portion of the spinal cord
(Cazalets et al. 1995
; Cowley and Schmidt
1997
), it was necessary to investigate whether well coordinated
locomotor patterns could equally be induced by bath application of
excitatory agents to the caudal segments only. An example is shown in
Fig. 1 (top right): caudal application of 10 µM 5-HT and 2 µM NMDA induced oscillations characterized by 2.8 ± 0.9 s
period, alternating between left/right and
flexor-related/extensor-related motor pools. In six of eight
preparations, a combination of 5-HT and NMDA applied to caudal segments
induced alternating oscillations in all four recorded VRs. It is known
that application of high K+ concentrations can
induce locomotor-like patterns in the entire spinal cord (Bracci
et al. 1998
); it was thus tested whether a K+-elicited excitation restricted to caudal
segments could also evoke alternating activity spreading to rostral
segments. Figure 1 (bottom left) shows that well coordinated
locomotor activity could be induced by application of 8.5 mM
K+ (4.2 ± 1.3 s period). Similar
results were observed in three of five preparations. It is worth noting
that rostral drug applications induced locomotor patterns in all
preparations tested and over a wide range of concentrations. On the
contrary, caudal applications induced persistent patterns within a very
narrow range of concentrations (2.5 µM 5-HT, 1 µM NMDA or 0.5 mM
K+ for each preparation) and in some cases failed
to elicit any rhythmic activity, thus confirming the greater
sensitivity of rostral segments to locomotion-inducing agents
(Kjaerulff and Kiehn 1996
). Further investigations were
undertaken to clarify whether any form of widespread excitation of the
caudal network by treatments known to induce epileptiform activity
could trigger episodes of locomotor activity in the rostral segments.
To this end Mg2+-free Krebs solution was
superfused via the caudal bath in six preparations.
Mg2+-free solution induced paroxysmal bursts of
activity lasting 1-10 s and with irregular frequency. At steady state
(>20 min from removal of Mg2+) the onset of each
burst was invariably synchronous among the four recorded roots (see
arrow in Fig. 1, bottom right). For a small (15%)
percentage of the analyzed events only, L2 VR
records were asynchronous. Such a phenomenon was observed occasionally during longer bursts (>5 s duration) and cannot be related to locomotor-like activity, because it lacked any rhythmicity. It is
possible that the strong excitatory input received by the rostral segments might have transiently activated crossed inhibitory
connections that prevented contralateral pools of motoneurons from
firing simultaneously.
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Further tests were performed by applying 50 µM 4-aminopyridine (4-AP) to the caudal bath: 4-AP induced an irregular bursting activity in VRs (not shown), characterized by frequent events of short-duration (between 1 and 2 s). In the three preparations tested, such bursts were always synchronous among the four recorded VRs and never triggered any alternating oscillations.
In summary, alternating oscillations could be detected in rostral segments following activation of caudal ones by agents that are commonly used to induce fictive locomotion, such as 5-HT, NMDA, or high K+ solutions.
Bursting activity in the caudal spinal cord propagated to the rostral part and triggered well-coordinated locomotor episodes
Because caudal application of excitatory agents (5-HT, NMDA, or high K+) could induce fictive locomotion, it was tested whether rostral locomotor episodes could also be triggered by spontaneous bursting resulting from pharmacological block of synaptic inhibition in caudal segments. The working hypothesis was that prolonged depolarization during each burst could spread rostrally to activate the portion of CPG in which inhibitory synapses were still operational.
In all these experiments, strychnine and bicuculline were applied at
saturating concentrations [respectively, 1 µM and 20 µM
(Bracci et al. 1996b)] via the caudal bath while
recordings were obtained from L5 and
L2 VRs. The early period (10-15 min) of
strychnine and bicuculline application is characterized by random
bursts that gradually turn into regular activity (Bracci et al.
1996a
), which in the present study had an average period of
34 ± 12 s and an average burst duration of 16 ± 8 s. Figure 2A shows an early
single burst recorded from L5 VRs (top pair of traces) characterized by prolonged baseline deflection and tonic firing. This burst could be synchronously detected in the rostral
L2 segments also (bottom pair of
traces in Fig. 2A) and was accompanied by alternating
oscillations lasting ~25 s. The period (3.2 s on average) of such
oscillations was within the range of typical locomotor frequencies and
the phase shift between left and right L2 VRs was
171 ± 22°, confirming their alternation. In 42/42 preparations
during the early phase of application of strychnine and bicuculline,
long-lasting (20-30 s) episodes of alternating activity were observed
in the rostral segments in correspondence with the caudal bursts, thus
demonstrating that disinhibition-activated caudal networks could
invariably trigger the rostral locomotor CPG.
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Figure 2B (different preparation from Fig. 2A)
represents, in the presence of caudal strychnine and bicuculline,
steady state bursting intracellularly recorded from a rostral
L2 motoneuron (top trace), two rostral
VRs (L1 and L2) and one
caudal L5 VR. In this example all extracellular
records were AC-coupled. Bursts in L5 VR
consisted of sustained excitation whereas lL2 and
rL1 VR activity was characterized by alternating
firing (usually no more then 2 or 3 cycles, with period ranging between
3 and 5 s). Firing in rL1 VR was always
synchronous with voltage oscillations of the ipsilateral
rL2 motoneuron (top record in Fig.
2B). The top trace of Fig. 2B depicts
the intracellularly recorded bursts (22 ± 4 mV amplitude and
~150-ms rise time from baseline to peak). During the interburst
interval, depolarizing events (that occasionally brought the neuron
above threshold for firing) due to ongoing spontaneous synaptic
activity frequently appeared. Motoneuron membrane potential (78 mV)
was not changed by application of strychnine and bicuculline (similar
results were obtained in 23 cells from 17 different preparations). For
each burst membrane potential oscillations (5- to 10-mV amplitude) were
accompanied by an increase in firing during the rising and top phase of
each oscillation. This phenomenon is depicted with expanded time scale in Fig. 2C. Extracellular DC coupled records show that
motoneuronal oscillations (top record) were out of phase
with the contralateral lL2 VR and in phase with
the ipsilateral rL1 VR. In 27/42 preparations, such oscillatory episodes were consistently observed during the whole
period of application, whereas in the remaining 15 preparations, alternating episodes were observed during early bursting, and only
occasionally (but still time locked with the occurrence of a burst
episode) after the disinhibited rhythmic activity reached steady state.
Simultaneous induction of disinhibited rhythm and fictive locomotion
A burst of activity in the caudal network could propagate to rostral segments and transiently activate the CPG for locomotion, suggesting that caudal commands could drive the main rostral CPG. Did this sort of entrainment preclude activation of the rostral CPG by the usual locomotor agents applied locally? This issue was tested in a series of experiments.
A locomotor pattern was induced by rostral application of 5 µM 5-HT and 4 µM NMDA and consisted of alternating activity clearly detected in rostral and caudal VRs (Fig. 3A). Phase relationships (open circles for 5-HT and NMDA treatment) were calculated for pairs of homolateral VRs (left or right L2-L5; see bottom 2 polar plots in Fig. 4) and contralateral VRs (l, rL2 and l, rL5; see top polar plots in Fig. 4). Each dot represents for the same preparation, the phase shift of 20-25 randomly chosen cycles recorded for the indicated pair of VRs (see METHODS). As data points were clustered around 180°, it is apparent that there was sustained alternation of activity between the indicated root pairs.
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Application of strychnine and bicuculline to the caudal bath (Fig. 3B) induced bursting activity in L5 VRs as shown by the large and persistent depolarizations with superimposed firing (Fig. 3B, top 2 traces). Such a bursting activity could also be detected in the rostral L2 VRs (Fig. 3B, bottom 2 traces) as smaller amplitude sustained depolarizations synchronous with L5 bursts and intermingled with the ongoing fictive locomotor pattern due to the continuous presence of 5-HT and NMDA. During the plateau phase of each burst in all four roots, fictive locomotion was absent. When it resumed, it maintained its expected alternation between L2 VRs (due to intact inhibitory circuitry), but, rather unexpectedly, alternation also appeared between L5 VRs despite the presence of strychnine and bicuculline. This observation is clearly shown in Fig. 3C in which the four roots patterns are recorded on a faster time base. The fact that the L5 segments possessed an alternating pattern in the presence of pharmacological block of inhibition suggested that this activity was driven by the rostral segments in which inhibition was still functional. Further proof for this proposal was provided by the observation that alternation was also maintained along the rostrocaudal axis between the homolateral pairs of L2-L5.
Phase relationship data for the activity present during caudal strychnine and bicuculline application plus rostral 5-HT and NMDA application are shown as filled circles in Fig. 4. Note that in this preparation, like in the other 41 tested, the phase values between contralateral L2 VRs completely overlapped those found in the absence of strychnine and bicuculline (compare filled and open circles, respectively). A similar overlap of phase values was observed for the other three pairs recorded in this experiment. Such oscillations were clearly detectable in L5 VRs in 25/42 preparations treated with 5-HT and/or NMDA. In 19/25 preparations, L5 oscillations were out of phase with homolateral L2 activity during fictive locomotion and so remained after caudal application of strychnine and bicuculline. In 6/25 spinal cords the phase between homolateral L2-L5 VRs was converted from alternating to synchronous after application of strychnine and bicuculline to the caudal bath. In the same six experiments rL2-lL2 and rL5-lL5 phases remained, however, in alternation (175 ± 21° and 169 ± 18° for l, rL2; 186 ± 26° and 195 ± 19° for l, r L5 for data before and after strychnine and bicuculline, respectively).
In summary then, in the majority of cases, alternation between VR activity during chemically induced locomotion was preserved between L2 and L5 VRs, despite pharmacological block of synaptic inhibition in caudal segments.
Midsagittal lesion and partitioned spinal cord configuration
The novel observation of clearly alternating oscillations in the
caudal spinal cord, despite the presence of saturating concentrations of strychnine and bicuculline, raised the possibility that caudal alternation at L5 segmental level was due to
inhibitory left-right synaptic transmission mediated by receptors
insensitive to these convulsants. To test this hypothesis, we performed
a midsagittal lesion (along the rostrocaudal axis) to bisect completely
the spinal segments located in the caudal bath (see dashed line in Fig.
5C). Figure 5A
shows the control locomotor pattern induced by rostral application of 5 µM 5-HT and 4 µM NMDA (3.2 ± 0.3 s period). Following
wash out of 5-HT and NMDA, caudal application of strychnine and
bicuculline elicited bursting activity that could be detected in the
four recorded VRs (not shown). Steady-state bursting induced by
strychnine and bicuculline was characterized by a period of 32 ± 8 s. Each burst comprised oscillations that were fast (~200 ms
period), synchronous, and occurred after a 20- to 40-min application of
strychnine and bicuculline as previously described (Bracci et
al. 1996a). In the early stage of strychnine and bicuculline
application, the initial epileptiform activity and the subsequent
gradual organization of spontaneous rhythmicity prevented the detection
of intraburst oscillations. Such events are clearly seen in the traces
from L5 VRs (Fig. 5B; steady-state strychnine and bicuculline application) and are thus distinct from the
fictive locomotor oscillations not only for their period but also for
the lack of alternation.
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When the same concentrations of 5-HT and NMDA were superfused via the
rostral bath while retaining strychnine and bicuculline in the caudal
one, fictive locomotion-like, alternating oscillations were detected
from L2 VRs as well as from
L5 VRs (Fig. 5B). The period of such
oscillations (3.6 ± 0.7 s) was remarkably similar to that
observed in control. This oscillatory activity was interrupted whenever
a burst occurred and was restored during burst decay. The phase of this
oscillatory activity calculated for contralateral or homolateral VRs
did not significantly change during rostral application of 5-HT and
NMDA before () and after (
) application of strychnine and
bicuculline to the caudal bath (Fig. 6).
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After making a midsagittal lesion from conus medullaris to the L4 segment, rostral application of 5-HT and NMDA (same concentration as in control) could still elicit a stable alternating pattern, with a period of 3.8 ± 0.5 s, as shown in Fig. 5D. Alternation in activity was preserved in L2 VRs, as well as in the hemisected L5 segment, thus confirming that in this condition alternation was driven from rostral segments to caudal ones. Open squares in Fig. 6 represent the phase relationship among the four recorded VRs: no significant phase difference was observed when compared with values obtained before lesioning.
Synergy between caudal bursts and chemical activation of the rostral segments
An interesting question was whether caudal bursts could trigger
rostral locomotor activity when the concentration of chemical agents in
the rostral bath was subthreshold for fictive locomotion. This type of
facilitation could in fact allude to a common network mechanism
responsible for disinhibited bursting and locomotor patterns. For this
purpose we applied increasing concentrations of NMDA to the rostral
compartment, while strychnine and bicuculline were applied to the
caudal end. Single bursts in the presence of different concentrations
of NMDA are shown in Fig. 7. With control
solution in the rostral bath, and with strychnine and bicuculline in
the caudal bath (Fig. 7, top left), alternating activity was
virtually absent. When 1 µM NMDA was applied rostrally (Fig. 7,
top right), alternating oscillatory activity was apparent during the entire double burst in the rostral VRs and then stopped. As
the NMDA concentration was increased to 2 µM (Fig. 7, bottom left), alternating oscillations appeared more pronounced, started during the burst, and persisted after the burst for ~15 s.
Application of 3 µM NMDA (Fig. 7, bottom right) induced a
regular pattern of rostral alternating activity that was suppressed by
the onset of a burst and recovered during its decay phase, persisting
until a new burst episode occurred. Similar results were observed in six different preparations. From these records it appears that increasing concentrations of NMDA were also associated with a smaller
amplitude of the rostral bursts elicited by strychnine and bicuculline.
The origin of this reduction in burst amplitude when NMDA (with or
without 5-HT) was applied was explored with intracellular recordings
from L2 motoneurons (n = 8).
Burst peak amplitude, which in control conditions was 41 ± 6 mV,
in the presence of NMDA and/or 5-HT was significantly reduced (28 ± 7 mV; P < 0.01 ANOVA test), whereas it was restored
to a value not significantly different from control (38 ± 6 mV)
by injection of negative DC current (n = 8 cells). In
these tests the average depolarization induced by NMDA and/or 5-HT was
23 ± 7 mV. The observation that burst amplitude could return to
control value by simply repolarizing the motoneuron suggests that it
was mainly due to the depolarizing action of 5-HT and NMDA, which
diminished the driving force for the burst currents that are known to
reverse at zero mV (Bracci et al. 1996a).
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The tight interplay between caudally originated bursts and rostrally elicited excitation by NMDA suggests a common rostral target for disinhibited bursting signals and for NMDA, namely the locomotor CPG.
Within a narrow (0.5 µM) window of NMDA concentrations (3/6 preparations), it was possible to observe persistent locomotor oscillations together with caudally induced bursts as exemplified in Fig. 8A in which four simultaneous recordings during caudal application of strychnine and bicuculline and rostral application of 4.5 µM NMDA are shown. L2 VRs displayed alternating oscillations (Fig. 8A) as long as 4.5 µM NMDA was superfused to the rostral bath. Time scale expansion of the underlined parts of these traces (arrow in the bottom part of Fig. 8A) indicated that the period of L2 VR alternating oscillations was 1.4 ± 0.3 s during bursts (Fig. 8A, bottom left) and increased to 3.1 ± 0.6 s immediately before a burst (Fig. 8A, bottom right). The period variation was quantified (Fig. 8B) by averaging the period of the alternating oscillations in L2 VRs occurring on top of each burst and comparing it with the period of oscillations occurring just before the onset of bursts. For 10 consecutive burst episodes, the average period was 1.7 ± 0.4 s during bursts and was significantly different from the average period (2.6 ± 0.5 s) before burst onset (P < 0.005).
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DISCUSSION |
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The present study provides a novel description of the intersegmental interaction between rhythmogenic networks in the neonatal rat spinal cord. Data obtained during simultaneous, focal activation of caudal or rostral networks, which expressed disinhibition-induced rhythm or fictive locomotion respectively, enabled us to propose a wiring diagram to account for the operation of burst generators in distinct segments of the spinal cord.
Selective activation of caudal and rostral rhythmogenic networks
Activation of either rostral or caudal portions of the spinal cord
by 5-HT and/or NMDA (or high K+ solution) induced
rhythmic alternating patterns that could be detected from both rostral
(flexor-related) and caudal (extensor-related) VRs. The present
findings thus support the conclusion that the CPG for locomotion is
distributed along the rostrocaudal axis (Kjaerulff and Kiehn
1996).
An important finding of the present study is that rostral as well as
caudal segments can be topically activated by 5-HT, NMDA, or high
K+ to produce locomotor patterns that spread to
the other portion of the spinal cord not exposed to such substances.
The present observations accord with data from Kjaerulff and
Kiehn (1996), who used a mixture of NMDA and 5-HT but differ
from those of Cazalets et al. (1995)
, who used a similar
mixture of these agents. It is worth noting that Cazalets et al.
(1995)
used a substantially higher concentration of the
locomotor pattern inducing drugs that were thus potentially liable to
produce large and sustained depolarization of spinal neurons detected
as tonic firing instead of rhythmic activity. Cowley and Schmidt
(1997)
have reported that, when applied caudally, 5-HT could
not induce locomotor patterns recorded from peripheral nerves, whereas
NMDA could elicit alternating patterns apparently unsuitable to
locomotion. This result led Cowley and Schmidt to conclude that the
locomotor CPG has a very rostral location at the thoracolumbar border.
Our observations of a caudally originated motor pattern with 5-HT plus
NMDA administration could not be explained simply on the basis of this
latter agent because the NMDA concentration was usually below rhythm
generating threshold (cf., for instance, Fig. 7) and was added just to
stabilize the alternating pattern (Kjaerulff and Kiehn
1996
). Our data therefore agree with those of Kjaerulff
and Kiehn (1996)
that the caudal areas are rhythmogenic,
although less so than the rostral one. Furthermore, we have noted that
very small changes in caudally applied drug concentrations were
necessary to elicit a stable pattern without precipitating it into
sustained tonic firing. Perhaps the experiments reported by
Cowley and Schmidt (1997)
did not rely on such a
critical dosage of locomotor agents, which were often applied in much
higher concentrations than the present ones.
The bidirectional propagation of locomotor patterns is compatible with
the theory of unit burst generators distributed along the lumbar spinal
cord (Grillner 1981). Hence the portions of the
locomotor network that were not exposed to the excitatory agents might
have received an excitatory drive (from the activated regions) that
triggered the locomotor program. Phase alternation of VR outputs would
be determined by the inhibitory/excitatory connections between burst
generators supplying different muscles.
Interaction between caudal and rostral networks
Application of strychnine and bicuculline to the caudal bath
invariably induced rhythmic bursting activity in all preparations tested. Onset and steady-state activity recorded from
L5 VRs were similar to those observed during
block of inhibition in the entire spinal cord (Bracci et al.
1996a). Individual bursts were usually characterized by a
plateau phase, followed by several high-frequency (5-10 Hz) intraburst
oscillations synchronously detected in L5 VRs. It
is noteworthy that, during application of strychnine and bicuculline to
the whole spinal cord, the interburst interval is characterized by a
complete absence of synaptic activity (Ballerini et al.
1997
; Bracci et al. 1996a
, 1997
).
On the contrary, in the present conditions spontaneous synaptic
potentials were frequently detected from L2
motoneurons during the interburst intervals, to indicate that the
ongoing synaptic activity remained intact in the rostral networks, as
strychnine and bicuculline were applied to the caudal end only.
The principal finding was, however, that bursts induced in caudal
segments could trigger alternating, locomotor-like oscillations in
L2 VRs. The excitation produced by each burst
apparently propagated to the more rostral segments to activate the
rostral unit burst generators. Although the input received by the
rostral units was presumably synchronous for the left and right
generators, the presence of intact inhibitory connections between
antagonist units might have allowed the CPG to respond to this signal
with left-right pattern alternation. For this phenomenon to occur, it
is necessary that the strength of the inhibitory left-right connections
prevailed over the synchronous excitatory input received from the
caudal segments (Kremer and Lev-Tov 1997) so that
antagonist motor pools never fired simultaneously. A rostral rhythmic
alternating pattern could not be evoked by caudally applied
Mg2+-free solution or 4-AP, suggesting that this
response was not a nonspecific stereotype of the spinal network to any
form of excitatory event.
The observation that disinhibition-induced bursting activity in caudal
segments could trigger locomotor episodes in the rostral ones suggests
that the network responsible for disinhibited rhythm impinges (directly
or indirectly) on the CPG for locomotion. This possibility was
confirmed by the experiments in which caudal application of strychnine
and bicuculline was followed by rostral application of
locomotion-inducing agents. In such conditions, persistent locomotor
oscillations in L2 VRs were always changed by the
occurrence of a burst through a repertoire that ranged from alteration
in period to full suppression. In fact, discrete changes in the
concentrations of the inducing agents in the rostral bath (i.e., of the
amount of ongoing excitation induced in the rostral segments) elicited alternating oscillations exclusively during the burst, or in its decay
phase, or in the interburst time, or even during and after bursts with related changes in periodicity. Suppression of
locomotor-like oscillations during bursts (a commonly observed
phenomenon) was likely due to excessive depolarization of
L2 motoneurons because excitatory synaptic inputs
coming from the caudal network summated with the direct depolarization
induced by 5-HT and/or NMDA as demonstrated with intracellular
recording. In addition, the input from the caudal network to the CPG
itself might have been so strong to inactivate it [as observed when
high doses of K+ or NMDA were applied to the
whole spinal cord (Bracci et al. 1998)].
Persistence of alternating oscillations despite application of strychnine and bicuculline
Application of strychnine and bicuculline to the caudal bath to
impair reciprocal inhibition between left and right motor pools
(Bracci et al. 1996b) should have canceled any phase
alternation of oscillations between the L5 VRs
during fictive locomotion rostrally induced by 5-HT and NMDA. On the
contrary, in the majority of preparations tested, locomotor-like
oscillations characterized by left-right alternation were surprisingly
found to persist in L5 VRs. Such oscillations
were observed during the interburst intervals only, because the
occurrence of a disinhibited burst invariably occluded them.
The presence of alternating oscillations in spinal cord segments, in
which fast synaptic inhibition is demonstrably blocked (see
Bracci et al. 1996b; Rozzo et al. 1999
),
might suggest that at the same segmental level inhibition was mediated
by activation of receptors other than GABAA and
glycine ones. Possible candidates for this role might be
GABAB or GABAC receptors
(Rozzo et al. 1999
), 5-HT1
receptors (Beato and Nistri 1998
; Elliott and
Wallis 1992
), or metabotropic glutamate receptors, that are
known to depress synaptic transmission in many different systems
(Scanziani et al. 1997
; van den Pol et al.
1998
). If this were the case, regardless of the particular
transmitter system involved, alternation should have been disrupted
following midsagittal lesion of the caudal segments as each side of the
spinal cord would have become unable to produce reciprocal inhibition.
Contrary to this possibility, lesion experiments showed that the
alternating pattern remained unchanged despite full bisection of the
spinal cord from conus medullaris to
L4-L3 segments. It seems
thus likely that motor output alternation in the presence of strychnine
and bicuculline in a sagittally split segment could only originate from
segments unaffected by lesioning or pharmacological blockers of
inhibition, namely those rostrally located beyond the transverse barrier.
Wiring diagram for the locomotor CPG
The present observations add further complexity to existing
schemes of the spinal locomotor CPG (Cazalets et al.
1995; Cowley and Schmidt 1997
; Kjaerulff
and Kiehn 1997
) and require a new minimal wiring diagram (Fig.
9). The building blocks of the spinal CPG
remain as a series of unit burst generators (Grillner
1981
) distributed along the rostrocaudal axis (these are shown
as shaded squares in Fig. 9). The transverse barrier used in the
present experiments is shown to divide the serially arranged burst
generators into caudal and rostral ones. Motoneuronal pools are
depicted as separate gray circles because they are not an intrinsic
part of the CPG as they merely provide the motor output (programmed by
the CPG; note excitatory signals from unit generators to motoneurons) to muscles (Grillner 1981
). The scheme of Fig. 9 is
further simplified, for sake of clarity, by assuming just extensor
motor pools in the caudal portion and flexor motor pools in the rostral
one. Each burst generator supplies its contralateral equivalent and its
homolateral antagonist with weak excitatory and strong inhibitory connections (see Kremer and Lev-Tov 1997
). A scheme that
relies on serially arranged connections alone remains, however,
insufficient to explain why activation of certain unit generators can
recruit distant ones with appropriate phase lock between them and
between distant segments as indeed observed with the bidirectional
ability of 5-HT or NMDA to evoke fictive locomotion regardless of the lumbar region of their application. One possibility is to assume that a
strong excitatory signal from a given segment is conveyed through a
major interlinking pathway (or via relay interneurons) to adjacent
segments that would then be prompted to express their rhythmic output.
Phase interlocking between segments might then be due to reciprocal
inhibition. This hypothesis cannot, however, explain the typical
locomotor-like rhythm with standard phase alternation in rostral and
caudal segments despite the presence of strychnine and bicuculline
caudally. This phenomenon persisted even after longitudinal splitting
of the lower spinal cord. We are therefore inclined to believe that
burst generators in distinct segments were connected by strong, crossed
pathways as indicated in Fig. 9. Intersegmental cross linking would
therefore provide the excitatory connections necessary to support
rhythmic alternation in an area where inhibition was blocked. In
addition, the crossover of this pathway should have taken place above
the transverse barrier because lower segments surgically separated were
still driven in a fully alternating fashion despite the splitting
lesion and block of inhibition. The currently proposed circuit should
be viewed as an operational scheme to be tested with computer modeling to reconstruct the CPG operation and predict certain pattern
activities.
|
Is a single CPG network sufficient to generate locomotor patterns and disinhibited rhythms?
The still incomplete understanding of the identity of the
interneurons that make up the locomotor CPG precludes a clear answer to
this issue. Nevertheless, a number of observations concur to suggest
that the single network hypothesis is the simplest to account for the
experimental findings of the present (and other) studies. In
particular, the intrinsically slow periodicity of disinhibited bursting
is converted into a relatively fast (1-2 s) period (lacking
alternation) by typical locomotor agents like 5-HT or NMDA
(Bracci et al. 1996a,b
): such an identical sensitivity of both patterns is best explained by a single network arrangement dynamically modulated and wired to produce different rhythms and patterns. Lesion studies show that the disinhibited rhythm and the
locomotor pattern are similarly located in the anterior quadrant of the
spinal cord (Bracci et al. 1996b
; Kjaerulff and
Kiehn 1996
). In addition, both locomotor patterns and
disinhibited rhythms can be expressed by the same rostral or caudal
segments (Kjaerulff and Kiehn 1996
; M. Beato,
unpublished data and the present study). Furthermore, the strong
synergy between disinhibited rhythm and locomotor pattern demonstrated
by the present study (bursts could be the threshold crossing process to
elicit a locomotor pattern or to modulate its periodicity) is also
suggestive of a common CPG origin.
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ACKNOWLEDGMENTS |
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We thank Drs. Ole Kiehn and Ole Kjaerulff of the Panum Institute, Copenhagen, Denmark, for kindly demonstrating the transverse split bath arrangement used for the present study.
This work was supported by grants from the Ministero dell' Università e della Ricerca Scientifica e Tecnologica (co-finanziamento ricerca) and from the Istituto Nazionale Fisica della Materia.
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FOOTNOTES |
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Address for reprint requests: A. Nistri, Biophysics Sector and Istituto Nazionale di Fisica della Materia Unit, International School for Advanced Studies (SISSA), Via Beirut 4, 34014 Trieste, Italy.
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 20 April 1999; accepted in final form 18 June 1999.
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REFERENCES |
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