Department of Anatomy and Cell Biology, The Hebrew University Medical School, Jerusalem 91120, Israel
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ABSTRACT |
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Lev-Tov, A., I. Delvolvé, and E. Kremer. Sacrocaudal Afferents Induce Rhythmic Efferent Bursting in Isolated Spinal Cords of Neonatal Rats. J. Neurophysiol. 83: 888-894, 2000. The ability of mammalian spinal cords to generate rhythmic motor behavior in nonlimb moving segments was examined in isolated spinal cords of neonatal rats. Stimulation of sacrocaudal afferents (SCA) induced alternating left-right bursts in lumbosacral efferents and in tail muscles. On each side of the tail, flexors, extensors, and abductors were coactive during each cycle of activity. This rhythm originated mainly in the sacrocaudal region because it persisted in sacrocaudal segments after surgical removal of the thoracolumbar cord. Sacrocaudal commissural pathways were sufficient to maintain the left-right alternation of lumbar efferent bursts, because their timing was unaltered after a complete thoracolumbar hemisection. The lumbar rhythm originated in part from sacrocaudal activity ascending in lateral and ventrolateral funiculi, because efferent bursts in rostral lumbar segments were nearly abolished on a particular side by lesions of the lateral quadrant of the cord at the L4-L5 junction. Intracellular recordings from S2-S3 motoneurons, obtained during the rhythm, revealed the presence of phasic oscillations of membrane potential superimposed on a tonic depolarization. Bursts of spikes occurred on the depolarizing phases of the oscillation. Between these bursts the membrane input conductance increased, and hyperpolarizing drive potentials were revealed. The inhibitory drive and the decreased input resistance coincided with contralateral efferent bursts, suggesting that crossed pathways controlled it. Our studies indicate that pattern generators are not restricted to limb-moving spinal segments and suggest that regional specializations of pattern-generating circuitry and their associated interneurons are responsible for the different motor patterns produced by the mammalian spinal cord.
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INTRODUCTION |
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The study of rhythmic networks in the mammalian
spinal cord has focused on the limb-moving brachial and thoracolumbar
segments. By contrast, much less information is available concerning
the rhythmogenic capacity of the nonlimb moving segments. In
particular, it is not clear whether all spinal segments contain
rhythmogenic circuitry, like simple vertebrates such as the lamprey
(Cohen 1987; Cohen and Wallen 1980
;
Grillner and Matsushima 1991
; Hagevik and
McClellan 1994
) or if such networks are restricted to certain parts of the cord.
Studies of locomotion in neonatal rats suggested that the rhythmogenic
circuitry associated with hindlimb locomotion was localized to the
L1/L2 spinal segments
(Cazalets et al. 1995). Other studies, however, revealed
that caudal lumbar segments could also generate rhythmic activity
(Cowley and Schmidt 1997
; Kjaerulff and Kiehn 1996
; Kremer and Lev-Tov 1997
) but that rostral
lumbar segments have a higher "rhythmic capacity" than caudal
lumbar segments.
Motoneurons innervating the hindlimb musculature of the rat
(Nicolopoulos-Stournaras and Iles 1983) and mouse
(McHanwell and Biscoe 1981
) are localized to
L1-L5 segments. The
present work was aimed at studying the capacity of spinal cord regions
that do not contain hindlimb-moving motoneurons to produce motor
rhythms. We have identified a network, located in the sacrocaudal
segments, that generates rhythmic tail movements. We show that this
network shared some principles of action with the locomotor network and differs in several respects from it. Some of the preliminary findings appeared in an abstract (Lev-Tov and Kremer 1999
).
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METHODS |
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Preparation
Spinal cord preparations were isolated from postnatal day
3-8 (P3-P8) ether-anesthetized rats (see
Kremer and Lev-Tov 1997) with, or without an intact
tail. The cord (rostral T6 and down) was transferred to a
recording chamber and superfused continuously (10-15 ml/min) with an
oxygenated Krebs saline (composition in mM: 128 NaCl, 4 KCl, 2 CaCl2, 1 MgSO4, 1 NaH2PO4, 25 NaHCO3, and 30 glucose), at room temperature (24-26°C), pH 7.3.
Stimulation and recordings
Suction electrode recordings from ventral roots were performed
using a high gain AC amplifier at 0.1 Hz to 10 kHz. Sharp electrode (60-100 M, 3 M K+-acetate micropipettes)
intracellular recordings were obtained from
S2-S3 motoneurons impaled
from the ventral or ventrolateral aspect of the cord and identified by
the presence of antidromic spikes (e.g., Lev-Tov and Pinco
1992
). When required, spiking activity of impaled motoneurons
was blocked by addition of 200 mM QX-314 to the recording pipette.
Stimulus trains (5-100 pulses, 10 Hz) applied to the
S4-CA1 dorsal roots at 1.2-6 times threshold (T) induced the rhythm. Single-ended electromyographic (EMG)
recordings (100 Hz to 10 kHz) were obtained by 0.003-in. silver
wires inserted into the respective tail muscles.
Data acquisition and analysis
Data were continuously recorded using a high-speed (22-88 kHz) PCM recorder (Neurodata), filtered using high- and low-pass filters, and stored for subsequent off-line computer analyses.
Analyses of the phase values of the EMG data were done by
descriptive statistics of circular distribution (Zar
1984; e.g., Kjaerulff and Kiehn 1996
). The raw
phase values, the computed mean phase, and the measure r
that describes the concentration of phase values around the mean were
plotted on a circular scale (Fig. 2B). Multisample testing
of the angles (the Watson-Williams test) (Zar 1984
) was
performed to compare between the resultant mean phase values.
CHANGES IN INPUT RESISTANCE (RN) DURING THE RHYTHM. The amplitudes of voltage transients produced in sacrocaudal motoneurons by negative current steps (excluding those obtained during the stimulus train) were normalized with respect to the mean prestimulus control calculated from the last 20 transients produced before each stimulus train. The means of the normalized RN values during the contra- and ipsilateral efferent bursts were calculated for each run (3 runs per cell). Statistical analysis was done by the use of a two-tailed t-test.
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RESULTS |
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Figure 1 shows that mechanical stimulation of the tail in hindlimb/tail-spinal cord preparations of the neonatal rat (Fig. 1A, left) induced alternating left-right bursts in pairs of lumbar (L2) and sacral (S2) ventral roots (Fig. 1A, top 2 pairs of traces). The alternating pattern of the L2 efferent activity was partially masked by a continuous firing, but became clearer when the same data were band-pass filtered at 0.1-200 Hz (Fig. 1A, bottom set of traces).
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Figure 1B shows that an alternating rhythm, similar to the one induced by tail pinch, could be evoked in L3 and S2 efferents by 10-pulse 10-Hz trains applied to the right S4 dorsal roots at 2.2T (top panel). The alternating left-right bursts persisted in the S2 efferents after removal of the thoracolumbar cord by transection at the L6-S1 junction (Fig. 1B, bottom panel). After the transection, the duration of the cycles was significantly decreased from 2.16 ± 0.34 s (mean ± SD; n = 27) before the cut to 1.15 ± 0.24 s (n = 23) after it, P < 0.00001. Similar changes were obtained in four additional experiments. Moreover, sacrocaudal rhythm similar to the one produced in the isolated sacrocaudal cord by electrical stimulation could also be induced in the isolated tail-sacrocaudal cord preparation, by a tail pinch (not shown).
Motoneurons innervating the hindlimb musculature of the rat
(Nicolopoulos-Stournaras and Iles 1983) and mouse
(McHanwell and Biscoe 1981
) are localized to
L1-L5 segments. Motoneurons innervating the
striated pelvic muscles are found mainly in L6 motoneurons (Schroder 1980
). Few of those motoneurons can also be
found in S1. Most rat motoneurons in S1, and
virtually all the motoneurons in S2-CA3 segments are known
to innervate the tail muscles (Masson et al. 1991
;
Ritz et al. 1992
). Stimulation of sacrocaudal afferents (SCA) in tail-spinal cord preparations initiated ventral flexion that
was followed by left-right abductions of the tail (not shown). The
arrangement of tail muscles around the CA1 vertebra (e.g., Brink
and Pfaff 1980
) is shown in Fig.
2A (left,
ventral side up). EMG recordings (6 experiments) were obtained from
flexor caudae longus (FCL), extensor caudae lateralis (ECL), and
abductor caudae dorsalis (ACD). Figure 2A shows EMG
recordings from the left and right FCL and ECL (middle
panel) and from FCL and ACD (Fig. 2A, right
panel). Stimulation (10-pulse, 10-Hz trains, bars) of the right
S4 dorsal root in these two experiments induced an alternating activation of the left and right tail muscles, and synchronous activation of flexor, abductor, and extensor muscles on
each side of the tail. Analyses of data obtained from four additional
experiments revealed that the rhythmic bursts of the left FCL, ECL, or
ADC lagged by a half cycle the bursts from the same muscles on the
right [phase lag = 0.46, phase concentration (r) = 0.88, n = 219; Fig.
2B, Left-Right]. This phase lag differed significantly
(0.0002 < P < 0.0005; Watson-Williams test)
(see Zar 1984
), from that observed between the EMG
bursts of the left FCL, ECL, and ADC (phase lag = 0.98, phase
concentration = 0.95, n = 93, Fig.
2B, Left), or between those of the right FCL, ECL, and
ADC (phase lag = 0.98, phase concentration = 0.9, n = 98, Fig. 2B, Right).
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In Fig. 1 we showed that SCA activation produced alternating left-right bursts in both the lumbar and sacral cord. Surgical manipulations were performed to examine whether the thoracolumbar locomotion generator and its associated commissural circuitry were responsible for this lumbar rhythm. Figure 3 shows that the alternating efferent bursts produced in L2 and S2 efferents (A) were not perturbed after the entire thoracolumbar cord was midsagittally split (B). The phase lag between the left and right L2 efferent bursts was 0.49, with phase concentration measure (r) of 0.96 (n = 11 bursts) in the intact preparation and 0.48, with r of 0.94 (n = 11 bursts) in the split preparations. Similar results were obtained in three additional experiments. These findings showed that the sacrocaudal commissural connectivity was sufficient to maintain the alternating efferent bursts in the split thoracolumbar cord and suggest that the lumbar motoneurons are driven by rostrally projecting sacrocaudal interneurons in response to stimulation of sacrocaudal afferents.
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To test this hypothesis directly, and to establish where the axons of the rostrally projecting neurons were located, we performed lesions of the lateral white matter at L4-L5 and established the effects on lumbar and sacral efferent activity (Fig. 4). Figure 4A shows a rhythm produced by a 100-pulse 10-Hz train applied to the right CA1 dorsal root at 1.5T, in which the ventral root potential oscillations were in phase at the sacral and lumbar levels, on each side of the cord. When we sectioned the right lateral quadrant of the cord at L4-L5 junction, the rhythmic slow potentials produced in the right L2 ventral root were nearly abolished (Fig. 4B, R-L2). The same effects were found in a total of four experiments and were similar when the left or right CA1 dorsal root was stimulated to induce the rhythm. With the exception of a shortening of the cycle time, the lesion had no measurable effect on the activity in the left ventral root, or on the sacral recordings. In the same preparation, a subsequent cut of the left lateral quadrant of the cord, substantially attenuated (but did not completely block) the lumbar rhythm on the left side, but did not alter the sacral efferent bursts (Fig. 4C). These results confirm the idea that the lumbar activity can be driven by the sacrocaudal cord, through axons traveling in the lateral and ventrolateral white matter funiculi.
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To investigate the synaptic basis for the rhythmic activation of tail
muscles, we obtained intracellular recordings from 32 S2-S3 motoneurons in 11 experiments. Figure
5A (top
panel) illustrates one recording from a motoneuron located in
the right S2 segment, together with the left and right
S2 ventral root recordings. Stimulation of the right CA1
dorsal root produced voltage oscillations superimposed on a prolonged
depolarizing potential. The peaks and troughs of the oscillations were
in phase respectively with the ipsi- and contralateral efferent bursts.
To establish whether the troughs were mediated by active inhibition,
the membrane potential was depolarized to 60 mV, by a continuous
injection of current. This procedure revealed the hyperpolarizing
component of the mixed excitatory postsynaptic potential/inhibitory
postsynaptic potential (EPSP/IPSP) evoked in the motoneuron by single
pulse stimulation of the ipsi- or contralateral dorsal root (CA1) at
1.1T (see inset in Fig.
5A). Short train stimulation of the CA1 dorsal root at 6T under this depolarized condition elicited continuous
firing in the cell, interrupted by clear hyperpolarizing shifts in
membrane potential (Fig. 5, B and C).
These hyperpolarizations progressively declined in amplitude throughout
the rhythmic episode (Fig. 5C).
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If the hyperpolarizations were mediated by inhibitory synaptic conductances (rather than disfacilitation), then they should be accompanied by a significant increase in membrane conductance. To test this possibility, we monitored the conductance changes during the rhythm by measuring the amplitude of voltage transients produced by injection of hyperpolarizing current steps before, and after SCA stimulation (measured in 8 S2 motoneurons in 5 different experiments). Figure 6 shows that the amplitude of these voltage transients was significantly attenuated (1-way ANOVA followed by Tukey method, P < 0.00001) from a prestimulus control value of 8.4 ± 0.3 (n = 20), to 4 ± 0.9 mV (n = 12) during contralateral efferent bursts, and then recovered to 7.3 ± 2 mV (n = 9) during the ipsilateral bursts (heavy bars). Quantitative analyses of the data obtained from the eight-recorded cells (see METHODS) revealed that RN decreased during the contralateral efferent bursts to 60.5 ± 13% of its mean prestimulus control level, and then recovered to 100.4 ± 9.8% of the control during the ipsilateral bursts. The difference between these normalized RN means was statistically significant (2-tailed t-test, P < 0.00001). Thus these findings support the idea that the hyperpolarizing phases of the rhythmic drive are mediated by synaptic inhibition, and that a substantial part of this input is distributed over the somatic membrane.
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DISCUSSION |
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Sacrocaudal networks generate rhythmic tail movements
In the present study we have described a spinal neural network
that is capable of generating rhythmic tail movements. This network is
located mainly in the sacrocaudal spinal cord, and it could be
activated by SCA in the isolated sacrocaudal cord following surgical
removal of the thoracolumbar segments. Thus the ability to generate a
coordinated motor rhythm is not unique to limb-moving spinal cord
segments. The rhythm induced by mechanical stimulation of the tail or
stimulation of SCA is expressed as a prominent tail flexion followed by
rhythmic left-right abduction. Therefore an engagement of the flexion
withdrawal reflex is suggested. In this respect, the sacrocaudal rhythm
resembled the flexor-reflex afferents (FRA)-induced efferent bursts in
DOPA-nialamide cat preparations (Jankowska et al.
1967a,b
; Lundberg 1979
; also see Hultborn
et al. 1998
) and the spontaneous rhythm appearing under the
same pharmacological conditions in muscle nerves of the cat's tail
(Wada et al. 1996
). The ability to generate the
sacrocaudal rhythm in our experiments in the absence of drugs may
reflect the higher excitability of these networks in the developing
spinal cord.
Functional organization of rhythmic tail movements and comparison to locomotion
PHASIC EXCITATION AND INHIBITION.
The locomotor drive in cat (Orsal et al. 1986;
Pratt and Jordan 1987
; Shefchyk and Jordan
1985
) and neonatal rat (Hochman and Schmidt
1998
) motoneurons is believed to originate from alternating phasic excitation and inhibition. Our studies showed that SCA stimulation induced a prolonged depolarization with superimposed membrane potential oscillations. Bursts of spikes occurred on the peaks
of the oscillations and hyperpolarizations developed between them.
These hyperpolarizations were accompanied by a large (40%) reduction
in input resistance consistent with phasic activation of inhibitory
synaptic conductances. Because the oscillations were superimposed on a
tonic depolarization, it is not clear whether their peaks are due to
phasic excitation of motoneurons. Although we cannot exclude its
presence, our results may also be explained by rhythmic inhibition
superimposed on a tonic excitatory drive. An alternative possibility,
that rhythmic excitation is superimposed on tonic inhibition, is
unlikely, given the increase of RN to the prestimulation level during ipsilateral efferent bursts.
CROSSED INHIBITION AND EXCITATION.
Determination of the phase between flexor and extensor efferent bursts
during FRA induced rhythms (Jankowska et al. 1967b; Lundberg 1979
; also see Baldissera et al.
1981
), and during fictive scratching in the cat (reviewed in
Gelfand et al. 1988
) and turtle (reviewed in
Stein et al. 1998
) involves activation of reciprocal inhibitory pathways. Interlimb coordination during the neurochemically induced locomotor rhythm in the neonatal rat has been attributed to
activation of crossed-inhibitory and excitatory pathways (Cowley and Schmidt 1997
; Kjaerulff and Kiehn 1997
;
Kremer and Lev-Tov 1997
). The rhythmic inhibition we
have recorded in sacral motoneurons is also likely to be regulated by
crossed pathways because the inhibitory potentials and the reduction in
RN were synchronized with
contralateral motoneuron discharge, and because another potential source for phasic inhibition, the mutual flexor-extensor inhibition described for limb-moving networks, is probably absent in our system
(see Fig. 2). Crossed-inhibition was also revealed from the
late inhibitory component of the mixed PSPs produced in S2 motoneuron by contralateral SCA stimulation (Fig. 5A,
inset). The early EPSP component of these PSPs reflected an
activation of crossed-excitatory pathways. Massive activation of these
pathways in lumbar and sacral segments of the rat (Kremer and
Lev-Tov 1997
) and mouse (Bonnot et al. 1998
)
cord could be obtained in the presence of the glycine receptor blocker strychnine.
PATTERN OF THE MOTOR OUTPUT. Despite these similarities between the motor output of limb and tail moving segments, we also found systematic differences between the activity patterns produced by the two types of networks. During locomotion, scratching and FRA induced rhythms flexor and extensor motoneurons alternate within a limb. In contrast, all of the muscles on one side of the tail (flexors, extensors, and abductors) are coactive during each cycle of rhythmic activity. Flexor/extensor alternation during locomotion is believed to be mediated mainly by inhibitory connections between interneuronal centers. Such inhibition is apparently not a characteristic of the circuitry controlling each side of the tail.
In summary, our work has shown that nonlimb-moving regions of the spinal cord contain networks capable of generating rhythmic motor output activity. This observation raises the possibility that rhythm-generating circuitry are distributed throughout the spinal cord, as in primitive vertebrates. We propose that the "generic" rhythmogenic networks in a particular region, together with their associated interneuronal circuitry, are specialized for the specific functions they control. In the limb-moving segments, these networks control locomotion, scratching, paw shakes, and other limb behaviors. In the sacrocaudal segments, the network can produce rhythmic movements of the tail.Rhythmic tail movements: functional implications
The rhythmic tail movements we described are a consequence of
hyperexcitability of spinal networks in the absence of supraspinal control. Yet these movements reveal the existence of neural networks that may produce specific motor behaviors under normal conditions. Rhythmic tail movements are used by various mammals as means of communication. Tail movements possibly assist in balancing the body
during climbing and locomotion. These latter functions require efficacious coupling between the limb and tail rhythmogenic networks. Strong mutual coupling between the thoracolumbar and sacrocaudal centers would indicate that they interact. In the present study we
demonstrated that the alternating left-right rhythm evoked by SCA in
the lumbar cord could be maintained in midsagittally split
thoracolumbar cords. We also showed that the lumbar rhythm was nearly
abolished on a particular side by lesions of the lateral quadrant of
the cord at the L4-L5
junction. These results suggest that the rhythm that developed in the
lumbar cord by stimulation of sacrocaudal afferents reached the lumbar
region by propriospinal axons, ascending in the lateral and
ventrolateral funiculi and driven by sacrocaudal activity. Moreover,
similar coupling has been found to exist also in the rostrocaudal
direction. The sacral cord was strongly driven by the thoracolumbar
generator during neurochemically induced locomotion (Kremer and
Lev-Tov 1997). Collectively these observations indicate the
presence of a strong mutual coupling between the locomotor and
tail-moving central pattern generators. As suggested above, this
coupling may be necessary to coordinate tail and limb movements for
balance during locomotion and other motor tasks (Bennett et al.
1999
; Wada and Shikaki 1999
; Walker et
al. 1998
).
Further studies
The sacrocaudal network described in the present work offers a simple and accessible model for future studies of neurogenesis of automatic movements in the mammalian spinal cord. Our studies of the SCA-induced rhythm are only at their initial stages. Recordings from lamina VII interneurons in the sacrocaudal cord of the neonatal rat (Lev-Tov, unpublished observations) revealed several groups of interneurons, some of which exhibited rhythmic bursting activity on SCA stimulation. Further studies are required to assess their identity, their possible relation to FRA pathways, and their role in generation of rhythmic movements.
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ACKNOWLEDGMENTS |
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The authors thank Dr. M. J. O'Donovan for helpful comments on the manuscript.
This work was supported by Grant 724/97 from the Israel Academy for Sciences and Humanities, Jerusalem, Israel, to A. Lev-Tov.
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FOOTNOTES |
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Address for reprint requests: A. Lev-Tov, Dept. of Anatomy and Cell Biology, The Hebrew University Medical School, PO Box 12272, Jerusalem 91120, Israel.
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 30 July 1999; accepted in final form 18 October 1999.
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REFERENCES |
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