Laboratory of Neural Control, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892-4455
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ABSTRACT |
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Burke, R. E., A. M. Degtyarenko, and E. S. Simon. Patterns of Locomotor Drive to Motoneurons and Last-Order Interneurons: Clues to the Structure of the CPG. J. Neurophysiol. 86: 447-462, 2001. We have examined the linkage between patterns of activity in several hindlimb motor pools and the modulation of oligosynaptic cutaneous reflex pathways during fictive locomotion in decerebrate unanesthetized cats to assess the notion that such linkages can shed light on the structure of the central pattern generator (CPG) for locomotion. We have concentrated attention on the cutaneous reflex pathways that project to the flexor digitorum longus (FDL) motor pool because of that muscle's unique variable behavior during normal and fictive locomotion in the cat. Differential locomotor control of last-order excitatory interneurons in pathways from low-threshold cutaneous afferents in the superficial peroneal and medial plantar afferents to FDL motoneurons is fully documented for the first time. The qualitative patterns of differential control are shown to remain the same whether the FDL muscle is active in early flexion, as usually found, or during the extension phase of fictive locomotion, which is less common during fictive stepping. The patterns of motor pool activity and of reflex pathway modulation indicate that the flexion phase of fictive locomotion has distinct early versus late components. Observations during "normal" and unusual patterns of fictive stepping suggest that some aspects of locomotor pattern formation can be separated from rhythm generation, implying that these two CPG functions may be embodied, at least in part, in distinct neural organizations. The results are discussed in relation to a provisional circuit diagram that could explain the experimental findings.
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INTRODUCTION |
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There is abundant evidence
that the spinal cord of vertebrates, from primitive fish to carnivores,
contains an autonomous central pattern generator (CPG) that can produce
coordinated patterns of motoneuron activation that resemble those
observed in actual locomotion (Grillner 1981;
Rossignol 1996
). A CPG for locomotion may also exist in
the human spinal cord, although it is more difficult to demonstrate
than in quadrupedal mammals (Calancie et al. 1994
; Dimitrijevic et al. 1998
). The hallmark for
identification of a CPG within the CNS is the production of
recognizable and reproducible patterns of rhythmic output in the
absence of instructive external drive from other parts of the CNS or
from peripheral sensory feedback.
Traditionally, the CPG for locomotion has been studied in terms of
rhythmic drive to motoneurons. However, it has been known for some time
that a variety of reflexes are modulated in amplitude and even reversed
in sign during different phases of the stepping cycle, both in animals
(Abraham et al. 1985; Andersson et al. 1978
; Buford and Smith 1993
; Duysens and
Pearson 1976
; Forssberg 1979
; Forssberg
et al. 1975
, 1977
) and man (Duysens et al. 1990
; Stein and Capaday 1988
; Van Wezel et al.
1997
). Intracellular recordings from motoneurons during fictive
locomotion have provided clear evidence that the locomotor CPG exerts
powerful control of transmission through reflex pathways as assessed by
phasic modulation of synaptic potentials (Andersson et al.
1978
; Schomburg and Behrends 1978a
,b
).
In a series of papers from this laboratory (Degtyarenko et al.
1996, 1998a
,b
; Fleshman et al. 1984
;
Floeter et al. 1993
; Gossard et al. 1996
;
Moschovakis et al. 1991
; Schmidt et al.
1988
), the control of cutaneous and muscle afferent reflex
pathways during fictive locomotion was used as a tool to investigate
the organization of spinal last-order interneurons that project to
particular motor nuclei (reviewed in Burke 1999
). This
work concentrated on synaptic pathways that project to the flexor
digitorum longus (FDL) muscle because its motoneurons exhibit unique
patterns of activity during locomotion in normal cats
(Carlson-Kuhta et al. 1998
; O'Donovan et al.
1982
; Smith et al. 1998
; Trank and Smith
1996
). During unperturbed stepping, the FDL usually exhibits
little or no activity during the stance phase but fires a brief,
vigorous burst around the time of foot lift-off. This is quite
different from the activity in FDL's close, albeit not exact,
mechanical synergist flexor hallucis longus (FHL) (see Lawrence
et al. 1993
; Young et al. 1993
), which is active
throughout the stance phase of stepping and becomes silent just before
FDL bursts. The difference is particularly interesting because FDL and
FHL share mutual monosynaptic group Ia excitation (Fleshman et
al. 1984
), which is usually an indication of functional synergy
(Eccles et al. 1957
; Lloyd 1960
). The
distinction between FDL and FHL firing patterns persists during
backward walking in the cat, although in this case, FDL fires at the
transition into stance rather than into swing (Trank and Smith
1996
).
The present paper examines observations obtained largely from FDL
motoneurons during spontaneous and stimulation-evoked fictive locomotion with regard to what they suggest about the organization of
the locomotor CPG itself. A preliminary report has appeared in abstract
form (Burke et al. 1996).
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METHODS |
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The methods used for the present experiments have been described
in recent reports from this laboratory (Degtyarenko et al. 1996,
1998a
,b
; Gossard et al. 1996
; Moschovakis
et al. 1991
). Some of the results reported in the present paper
were extracted from data tapes made during this earlier work but not
previously published. We are grateful to Drs. Adonis Moschovakis,
Gerald N. Sholomenko, Mary Kay Floeter, and Jean-Pierre Gossard for
their participation in collecting these data. The experiments were
conducted in accordance with the "Principles of Laboratory Animal
Care" (National Institutes of Health Publication 86-23) and were
approved by the National Institute of Neurological Disorders and Stroke Committee on Animal Care and Use.
Briefly, 50 adult female cats (2.5-4.0 kg) were used. Halothane anesthesia was induced by mask and maintained (1-2% in air or oxygen) via a tracheal cannula during surgery. One common carotid artery was cannulated for blood pressure monitoring and the other was ligated. Intravenous catheters were placed in both cephalic veins for administration of norepinephrine and fluids as necessary to maintain blood pressure within physiological limits. The urinary bladder was catheterized. Rectal temperature was maintained near 38°C with a heating pad and lamp.
The following muscle nerves in the left hindlimb were cut and mounted on bipolar platinum wire electrodes for stimulation and recording: posterior biceps-semitendinosus (PBST), lateral gastrocnemius and soleus (LGS), medial gastrocnemius (MG), FDL, FHL, tibialis anterior (TA), and extensor digitorum longus (EDL). Cutaneous branches of the superficial peroneal (SP), medial plantar (MPL), and, in a few experiments, the saphenous (SAPH) nerves were freed but not cut; all were mounted on bipolar stimulating electrodes. Muscle nerves in the other limbs were not prepared for recording because the primary aim of these experiments was to examine ipsilateral reflex pathway modulation.
After a laminectomy exposing spinal segments L4-S1, the animals were transferred to a stereotaxic frame and skin flaps surrounding the spinal cord, and the hindlimb nerves were used to construct paraffin oil pools. Precollicular postmammillary decerebration was performed by removing all rostral brain tissue and cauterized vessels. Anesthesia was then discontinued. The animal was paralyzed with gallamine triethiodide (Flaxedil; 10 mg/kg supplemented every 40-60 min) and artificially ventilated to maintain expired CO2 near 4%.
Recording and stimulation
The cord dorsum potential (CDP) was recorded with a platinum
ball electrode placed near the dorsal root entry zone at the L6-L7 border. Stimulation
intensity to peripheral nerves was expressed in multiples of the
threshold for the most excitable fibers in the nerve (usually at twice
threshold or 2 × T). Intracellular recordings from motoneurons in
the L6-L7 segments were
made with glass micropipettes (1.0-2.0 µm tip diameter) filled with
2 M K+ acetate solution containing 26 mM QX314
(Alomone Laboratories, Jerusalem, Israel) to suppress sodium-dependent
action potentials (Frazier et al. 1970). Motoneurons
were identified by antidromic invasion from muscle nerve stimulation
during the several minute period required for spike blockade to occur.
To produce fictive locomotion, a monopolar tungsten electrode insulated
except at the tip was placed into the mesencephalic locomotor region
(MLR; nominal coordinates: P2, L4, HC 1), usually ipsilateral to the
side of recording. Constant-current (70-150 µA) biphasic, charge
balanced pulses (pulse duration, 0.2-0.5 ms separated by an equal
interval) were delivered in trains (12-30 Hz), referenced to a wire in
the neck muscles. The optimal position of the MLR electrode was
adjusted to produce rhythmic alternating activity in hindlimb muscle
nerves (fictive locomotion), which was usually accompanied by
distinctive CDP waves (Degtyarenko et al. 1998a
).
Data collection and analysis
Intracellular potential, CDP, and electroneurogram (ENG) activity in muscle nerves (usually LGS, FHL, FDL, TA or EDL, and PBST), were recorded with an eight-channel digital videotape recorder (Instrutech VR-100B; band-pass DC-9 kHz for the intracellular channel). Timing pulses that were synchronized with stimuli delivered to peripheral nerves and to the MLR were recorded on digital signal channels to permit selected synaptic potentials to be averaged during off-line data analysis.
Synaptic potentials were evoked in motoneurons during fictive
locomotion by alternately stimulating the SP and MPL nerves, usually at
a rate of 10 Hz each (Fig. 1) (see
Moschovakis et al. 1991 for detailed discussion of the
technique). The data from selected portions of the data tapes were
digitized off-line (10 kHz) using an Apple Macintosh PowerPC computer
and National Instruments NB-MIO-16 A/D board. Data collection and
analysis were done with "virtual instrument" programs written with
the LabView software package (National Instruments, Austin, TX). In
most cases, the phases of fictive locomotion were defined from the
digitized ENG data streams, which were rectified and smoothed using
decremental look-ahead exponential weighting with a time constant of
~34 ms. This algorithm provided a good match between the onsets and
offsets of raw ENG bursts and the smoothed waveforms.
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The program allowed ENG burst onsets and offsets to be determined either automatically by threshold crossing or manually when signal-to-noise ratios were low. The start of each flexion phase during fictive locomotion was taken as the onset of firing in the flexor motoneurons, FDL and/or PBST, and the offset of firing in extensor nerves (LGS and/or FHL) and ended with the termination of bursts in TA or EDL muscle nerves and the onset of extensor firing. The remaining time periods were defined as extension phases (e.g., Fig. 3). The flexion and extension phases were each subdivided into three equal time bins. Because of their variable durations, the time bins in the two phases usually did not have the same absolute durations.
The timing pulses associated with central and peripheral nerve stimuli
were used to trigger the computer to average together the intracellular
potentials and CDPs resulting from stimuli falling into the appropriate
locomotion phase bins (Degtyarenko et al. 1996;
Moschovakis et al. 1991
). Each average included at least eight sweeps and sweep numbers were usually >20. The MLR stimuli were
not synchronized to these pulses. When MLR stimulation produced significant postsynaptic potentials (PSPs), cutaneous PSPs were averaged only when they fell within an acceptance window that excluded
nearby MLR stimuli. The analysis program also allowed exclusion of
responses that produced action potentials (Degtyarenko et al.
1996
).
Central EPSP latencies were measured as the time between the peak of the first deflection of the CDP and the onset of the intracellular PSP (e.g., Fig. 4, B and D). This assumes that the excitatory PSP (EPSP) results from action of the most rapidly conducting afferents in the peripheral nerve.
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RESULTS |
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The aim of the present report is to discuss the implications of
interactions between cutaneous and muscle afferent reflex pathways and
the CPG for locomotion in the adult cat spinal cord. Some of the
material in this paper is drawn from experiments that have been
reported elsewhere in other contexts (Degtyarenko et al. 1996,
1998a
,b
; Gossard et al. 1996
). However, the
specific observations in this paper have not been published previously.
Locomotor modulation of cutaneous EPSP in FDL motoneurons
Previous publications from this laboratory have demonstrated
differential modulation of oligosynaptic EPSPs produced by
low-threshold afferents in the SP and MPL nerves in FDL motoneurons
during fictive locomotion in adult cats (Moschovakis et al.
1991; see also Degtyarenko et al. 1996
, 1998b
).
However, the earlier papers did not provide complete documentation of
the available material. Figure 1 presents summary records of averaged
SP EPSPs from 33 FDL motoneurons during extension and early flexion
phases of fictive locomotion (B and C), and EPSPs
during periods without fictive stepping in 12 of these cells
(A). Corresponding records of MPL EPSPs in 32 of the same
FDL cells are shown in D-F. With both inputs, EPSPs during extension phases resembled those at rest. However, the SP EPSPs during
early flexion (Fig. 1C) were notably larger and displayed shorter central latencies. In marked contrast, the MPL EPSPs during early flexion were markedly reduced (Fig. 1F).
The amplitudes and latencies of these EPSPs are shown in Fig.
2. The data in each graph are indexed
sequentially on the abscissae by increasing extension phase EPSP
amplitudes (A and C, ; measured at 2.5-ms
central latency, - - - in Fig. 1) or minimum central latencies
(B and D; see METHODS). The
amplitudes of SP and MPL EPSPs in the absence of fictive locomotion
("rest") were roughly the same as those found during the extension
phases of locomotion. However, the peak amplitudes of SP EPSPs at 2.5 ms during the flexion phase were considerably larger in most (25/33)
FDL cells (Fig. 2A). In contrast, the flexion phase MPL
EPSPs were either undetectable or much smaller than the extension phase
responses in all 28 FDL cells that exhibited any extensor phase EPSP.
Central latencies of SP EPSPs (Fig. 2B) were shorter during
flexion than in extension or at rest in most cells (28/33), with the
majority
2.0 ms (27/33). Over half of the sample of MPL EPSPs had
central latencies
2.0 ms at rest or during the extension phase, and
all but two FDL cells had MPL EPSPs with latencies
2.2 ms. It was not
possible to measure central latencies of MPL EPSPs during flexion.
Cutaneous EPSPs with central latencies
2.2 ms are assumed to be
disynaptic in the subsequent material (see DISCUSSION).
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Influence of afferent stimulation on FDL firing patterns
To define patterns of locomotor modulation of a reflex pathway it
is necessary to superimpose low-frequency repetitive stimulation of
pathway afferents during spontaneous or MLR-induced fictive locomotion
(Schmidt et al. 1988; Schomburg and Behrends
1978b
). Superimposing afferent input on locomotor rhythms can
change step-cycle durations as well as patterns of motoneuron activity,
especially with relatively high-frequency input (Fleshman et al.
1984
). Although we have not systematically analyzed the
possible alterations in the baseline "state" of the locomotor CPG
produced by our method of probing reflex pathways, considerable
experience with this method suggests that low-frequency, low-strength
cutaneous stimulation produces only subtle changes in the system.
Indeed, such changes are a major issue in this report.
In the example shown in Fig.
3A, with stimulation of the SP
nerve alone at 10 Hz superimposed on spontaneous locomotion, FDL fired
in short bursts during the first third of the flexion phase, accompanied by sharp depolarizing locomotor drive potentials (LDPs) (Jordan 1983) evident in the intracellular record from
an FDL motoneuron (top trace; arrows). In contrast, during
superimposed 10-Hz stimulation of the MPL nerve alone, the depolarizing
LDPs and the FDL bursts disappeared (open arrows), without changing the
basic locomotor rhythm. Mean step cycle durations were 2.5 ± 0.3 (SD) s during SP alone and 2.4 ± 0.6 s during MPL
alone stimulation. The hyperpolarizations of the FDL motoneuron during the last 2/3 of flexion in the two episodes were superimposable. Comparison of averaged hyperpolarizing pulses delivered to the motoneuron (5 nA, 10 Hz interleaved with nerve stimuli; downward deflections in the intracellular traces) showed that the cell input
resistance (RN) decreased during late
flexion (RN = 0.60 M
from
RN = 0.75 M
during extension),
indicating that the hyperpolarization resulted from active inhibition
of the FDL motoneurons coincident with TA motor pool firing. Similar
behavior was observed in several other animals.
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An analogous result was found in a different animal with SP stimulation at two intensities superimposed on spontaneous fictive stepping (Fig. 4). With 10 Hz SP stimulation at 1.5 × T, there were no depolarizing LDPs in the intracellular records from an FDL motoneuron, and no early flexion bursts in either FDL or PBST muscle nerves (open arrows). Nevertheless, the averaged SP EPSPs were clearly modulated, with maximum facilitation during F1 (Fig. 4B, heavy arrow) and minimum central latency of 2.2 ms. Increasing the stimulus strength to 2.0 × T produced depolarizing LDPs and bursting in both FDL and PBST nerves (Fig. 4C), with little evident alteration in the frequency of fictive stepping (the time base in A and C is the same). The EPSPs averaged with the higher stimulus intensity were larger and the minimum central latency decreased, by ~0.4 ms, to 1.8 ms (Fig. 4D; arrows). The same qualitative pattern of facilitation was evident at both SP stimulus intensities. The similarities in SP EPSP shapes and modulation suggest that the short-latency responses at 1.5 × T (~2.2 ms) were probably disynaptic (see DISCUSSION).
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Extensor phase activity in FDL
In addition to its stereotypical activation at the transition from
stance to swing during walking in intact cats (O'Donovan et al.
1982; Trank and Smith 1996
), FDL motoneurons can
also exhibit variable amounts of activity during the stance phase
(i.e., co-active with FHL) during perturbed step cycles and in some
other situations (O'Donovan et al. 1982
). Extensor
phase FDL activity can also occur during fictive locomotion
(Fleshman et al. 1984
; Moschovakis et al.
1991
), although we have been unable to find any set of conditions that produce it reliably. Nevertheless, some cases of stable
fictive stepping were encountered during which FDL was co-activated
with FHL during the extension phase during intracellular recording from
FDL motoneurons and stimulation of cutaneous afferents, allowing us to
evaluate the modulation of SP and MPL EPSPs in this state.
In the example shown in Fig. 5, during MLR-evoked fictive stepping with alternating SP and MPL stimulation (2 × T for each nerve) superimposed, the FDL ENG was co-active with LGS and FHL firing, without F1 bursts in the ENG (open arrows). The intracellular potential (FDL IC) showed only hyperpolarizing LDPs during F2 and F3, without F1 depolarizations (open arrows). However, Fig. 5B shows that the pattern of SP EPSP modulation was essentially the same as observed during bouts of fictive locomotion when FDL was active in F1 (cf. Fig. 4D). Unfortunately, the MPL nerve generated only small inhibitory PSPs (IPSPs) in this motoneuron during all phases of locomotion (not illustrated).
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The example in Fig. 6 illustrates a case in which the usual patterns of F1 FDL firing, depolarizing LDPs, and differential modulation of SP and MPL EPSPs were all present during relatively slow, spontaneous fictive locomotion (Fig. 6, A and B). However, when the MLR was stimulated, the stepping rate increased markedly and FDL was co-active with FHL during the extension phase. The F1 depolarization and FDL ENG bursts disappeared (Fig. 6C, open arrows). However, the pattern of modulation of SP and MPL EPSPs (D) were qualitatively similar to that in B, although there was less SP EPSP enhancement and less complete suppression of late MPL EPSPs during F2 and F3 than during spontaneous locomotion.
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Two distinct subphases during the flexion phase
The postsynaptic drive from the locomotor CPG to FDL motoneurons
changes dramatically from excitation to inhibition about one-third of
the way through the flexion phase in many examples of fictive stepping
(e.g., Figs. 3A and 4C). Although the FDL motor
pool can fire in F1 or during extension (and occasionally in both), we
have never observed FDL firing during mid- and late flexion during
fictive locomotion. The facilitation of disynaptic SP EPSPs during F1
usually disappears in F2 and F3 (Figs. 4, B and
D, and 9B) (see also Degtyarenko et al.
1996, their Fig. 9; Moschovakis et al. 1991
,
their Figs. 4 and 9), accompanied by progressive diminution of the
longer latency, trisynaptic EPSPs (Figs. 4-6). These observations
suggest the existence of distinct subphases in "early" versus
"late" flexion. We encountered one unusual animal that provided
additional evidence for these subphases.
The preparation illustrated in Fig. 7 exhibited spontaneous fictive locomotion with irregular step cycles during alternating stimulation of SP and MPL nerves at 10 Hz. The durations of FHL (extensor) phase bursts varied between cycles while the durations of EDL (mid- to late flexor) were uniformly brief. However, the most striking observation was that FDL bursts, in conjunction with PBST, were alternately long and short, without the brief F1 bursts characteristic of most examples of fictive locomotion (e.g., Figs. 4C and 6A). Remarkably, each of the flexor phases ended with uniformly short-duration bursts in EDL. There was no overlap between FDL bursts and firing in EDL (Fig. 7B). The simultaneous intracellular record from an FDL motoneuron (FDL IC) showed depolarizing LDPs during FDL firing with little evident hyperpolarization during EDL bursts. In fact, the membrane potential was most hyperpolarized during FHL activity.
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Figure 7, C and D, respectively, illustrate
modulation of SP and MPL EPSPs in the FDL motoneuron, averaged
separately during three periods of ENG activity: FDL, EDL, and FHL. The
SP EPSPs (C) were markedly enhanced during the periods when
FDL motoneurons were active (F[FDL]) but returned to the extensor
phase amplitude during the uniformly short bursts of EDL activity
(F[EDL]). The MPL EPSPs exhibited the usual flexion phase suppression
during EDL activity. However, during the F(FDL) phase, the initial EPSP component appeared unchanged from extension, but, surprisingly, there
was enhancement of a later, clearly trisynaptic component (C, arrow). Although the minimum central latencies of both
SP and MPL EPSPs were both slightly longer than 2.0 ms, we assume that
they were both disynaptic while the later components (C and D, heavy arrows) were trisynaptic (see
DISCUSSION). We have previously observed facilitation of
trisynaptic MPL EPSPs during FDL firing in unusual instances of
extensor phase FDL activity (Moschovakis et al. 1991,
their Figs. 9 and 10).
Stimulation of the SP nerve generates disynaptic IPSPs in EDL
motoneurons during early flexion in fictive locomotion
(Degtyarenko et al. 1996). Intracellular records from an
EDL motoneuron that exhibited this behavior in the same cat illustrated
in Fig. 7 are shown in Fig. 8. The SP
IPSPs in this EDL motoneuron were enhanced through the entire period of
flexion (Degtyarenko et al. 1996
, their Fig. 4). The
disynaptic MPL EPSP in this cell, which was not shown in that paper,
had a central latency of 1.8 ms. Quite unlike the MPL response in the
FDL motoneuron (Fig. 7D), the MPL EPSP in the EDL cell (Fig.
8) showed complete suppression during the FDL as well as EDL firing
periods, with no facilitation of the trisynaptic component (cf. Fig.
7D).
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Evidence for independent cycling of extensor and flexor CPG centers
Figure 9A illustrates
another unusual example of fictive locomotion in which the activity in
the extensor LGS exhibited sinusoidal waxing and waning activity with
~3 s between cycles. Uniformly short bursts of FDL, TA, and PBST
firing with the normal flexion phase sequencing were interjected into
this rhythm, skipping extensor cycles at the start of the recording and
later locked to the first half of the extensor cycles. These flexion
bursts were accompanied by early (F1) depolarizing LDPs and later
hyperpolarization in the FDL intracellular record (A, FDL
IC; ). In contrast to the rather bizarre and variable flexion bursts
shown in Fig. 7A, the flexion bursts in this example gave
the appearance of stereotyped events that were triggered in variable
phase relations with the extensor rhythm.
|
Averaged records of SP (B) and MPL EPSPs (C) were
obtained during the F1-F3 phases of flexion and during periods when
LGS activity was high versus low (Ext on and Ext off, respectively). The SP EPSPs exhibited the usual facilitation of disynaptic
(B, ) and trisynaptic components during F1 with
diminishing enhancement later in flexion. All of these responses were
larger than those during the extension phase, which showed only a small
difference depending on whether the LGS motor pool was active or not.
Suppression of MPL EPSPs was evident throughout flexion, but there may
have been some extensor phase facilitation of the trisynaptic component between LGS waves. Analogous observations of apparent independent cycling of extensor muscles with variable coupling to flexor bursts were made in two other cats.
Apparent independent cycling of flexor muscle pools is illustrated in
Fig. 10, obtained during MLR-evoked
fictive stepping during intracellular recording from an LGS motoneuron
(A, LGS IC) during interleaved stimulation of the SP nerve
and of the medial longitudinal fasciculus (MLF) (Floeter et al.
1993; Gossard et al. 1996a
). At the onset of MLR
stimulation, there was continuous activity in the recorded flexor ENGs,
but after ~40 s, the EDL neurogram began to exhibit the oscillations
evident in Fig. 10A with irregular, brief bursts in extensor
nerves. The intracellular potential in the recorded LGS motoneuron (LGS
IC) showed sinusoidal hyperpolarizations that were time-locked to the
EDL waves, suggesting that inhibitory interneurons that inhibit LGS
during flexion were also entrained. The PBST neurogram also showed
waves of activity that were about 180° out of phase with those in
EDL. This phase difference fits with other examples of fictive stepping
in which PBST begins to fire at the onset of the flexion phase, often
in concert with FDL (e.g., Figs. 3-6), and then wanes as EDL activity increases toward the end of flexion (e.g., Fig. 6A). The
short extensor bursts in the LGS and FHL nerves appeared to be
time-locked to the sinusoidal cycling of EDL but with variable numbers
of EDL cycles between them. Each extensor burst began in the middle of
the next expected wave of EDL firing, preceded by rapid, synchronized EDL bursts that were time locked to the MLR stimulation at ~14 Hz. As
in Fig. 9, these interjected extensor bursts did not appear to change
the EDL rhythm.
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Figure 10, B and C, illustrates averaged SP and
MLF EPSPs, respectively. Apparently di- and trisynaptic SP EPSPs were
generated in the LG motoneuron only when the LGS nerve was silent,
irrespective of whether EDL was active or not. They were completely
absent during the interjected extensor bursts (B, Ext). We
have observed similar modulation of oligosynaptic SP EPSPs in a
minority of triceps surae motoneurons (unpublished results).
Low-frequency stimulation of the MLF produces mono- and disynaptic
EPSPs in many types of hindlimb motoneurons. The disynaptic component
exhibits systematic enhancement during the stepping phase when the
motoneurons are active (Floeter et al. 1993;
Gossard et al. 1996
). Figure 10C illustrates
this extensor phase facilitation of disynaptic MLF EPSPs during the
interjected bursts of LGS and FHL activity. The disynaptic MLF EPSPs
were unchanged during the flexor cycling.
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DISCUSSION |
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The observations in this paper support the early surmise
(Burke and Fleshman 1986) that the patterns of
modulation of reflex pathways can provide useful clues to the structure
of the CPG for locomotion because they are as robust markers for
phase-related activity as are the activity patterns in different motor
pools. We have focused primarily on the FDL muscle because of its
unique behaviors during normal and fictive locomotion. In the
relatively few studies available in which FDL EMG activity in intact
cats has been studied in isolation (Abraham et al. 1985
;
Carlson-Kuhta et al. 1998
; Loeb 1993
;
O'Donovan et al. 1982
; Trank and Smith 1996
), FDL is normally activated in a brief burst at the
transition from late stance to early swing, entirely out of phase with
its mechanical synergist FHL. Activities of the two muscles are also mostly out of phase during actual (Carlson-Kuhta and Smith
1990
; O'Donovan et al. 1982
) and fictive
scratching (Degtyarenko et al. 1996
). Remarkably, this
behavior of the FDL motoneuron pool persists long after it
cross-reinnervates the soleus muscle (O'Donovan et al.
1985
). However, the FDL can also exhibit varying amounts of
activity during the stance phase and co-activation with FHL during
irregular stepping and in other actions in intact cats (O'Donovan et al. 1982
), as well as during fictive
locomotion (Figs. 5 and 6) (Fleshman et al. 1984
;
Moschovakis et al. 1991
).
The fact that these distinctive behaviors are found during
fictive locomotion in spinalized as well as decerebrate preparations (Moschovakis et al. 1991; Schmidt et al.
1988
) suggests that the underlying circuits are represented in
the spinal CPG for locomotion. The present material provides evidence
about the modulation of cutaneous reflex pathways during these
different FDL activity states. The data are used to develop a suggested
circuit diagram that speaks to the organization of the CPG as it
operates during fictive locomotion in the decerebrate cat. Although our
experimental design did not include recordings from the contralateral
hindlimb (see METHODS), the fact that the timing of
activity in FDL in relation to other motor pools in most of the present
work (Figs. 3, 4, and 6 and earlier references) resemble those found in
intact cats indicates that we are indeed studying fictive
"locomotion." As cited in INTRODUCTION, patterns of
reflex pathway modulation compatible with those observed in the present
work have been found in intact cats walking on treadmills. We assume
that the unusual examples shown in Figs. 7-10 also represent
locomotion in states of the system that reveal its internal details.
Reflex pathway length
Conclusions that can be drawn from reflex pathway modulation
during fictive locomotion are most secure when the pathway between peripheral afferents and the target motoneurons is disynaptic (i.e.,
when there is single layer of interposed interneurons) (see
Lundberg 1975). Evidence discussed elsewhere
(Degtyarenko et al. 1996
, 1998b
; Moschovakis et
al. 1991
) suggests that central latencies
2.0 ms indicate
disynaptic connectivity in cutaneous reflex pathways but that under
some conditions EPSPs with latencies
2.2 ms can be disynaptic, as
found for disynaptic group I EPSPs that are clearly disynaptic
(Degtyarenko et al. 1998b
; their Fig. 7).
The central latencies for the sample of SP EPSPs in Fig. 2B
ranged smoothly from 1.7 to almost 3 ms during the extension phase of
fictive locomotion and at rest (1.9-2.6 ms). However, during F1
facilitation, the latencies in 27/33 FDL motoneurons were 2.0 ms and
2.3 ms in all but one cell. Central latencies for MPL EPSPs during
the extension phase (Fig. 2D) increased smoothly from 1.6 to
~2.2 ms, with two examples
2.5 ms. The records of SP EPSPs in Fig.
4, B and D, show that simply increasing SP
stimulus intensity decreased central latencies of both early and later EPSP components by ~0.4 ms, from a minimum of 2.2-1.8 ms. The latter
is clearly disynaptic and it can be argued from the qualitative similarity of EPSP shapes at the two intensities that the early response at 1.5 × T was in fact disynaptic, and the later one trisynaptic. For these reasons, we regard central latencies
2.2 ms as
indicative of disynaptic connectivity for the SP and MPL pathways
discussed in this paper (e.g., including SP EPSPs in Figs. 4 and 7).
Locomotor CPG is an embedded system
Central pattern generators are sometimes depicted as autonomous
neural circuits, partly perhaps because the best known examples in
invertebrates can indeed function when completely isolated from the
rest of the organism's nervous system (e.g., Selverston et al.
1998). However, even invertebrate CPGs in situ receive information and may deliver feedback to the CNS in which they are
embedded (Selverston 1980
). Operation of a CPG can
continue in the absence of sensory input but it is clear that sensory
information modifies and controls many aspects of CPG output (e.g.,
Pearson et al. 1998
; Whelan 1996
). These
points are emphasized in Fig. 11, which
shows a generic CPG in the vertebrate spinal cord (within the dashed
rectangle) receiving primary afferent input and feedback from premotor
interneurons as well as descending control of both rhythm generation
and pattern formation functions. The arrows that inter-connect the
elements do not reflect specific circuits but are meant to signify the
complex interactions that are known to exist between spinal cord
interneurons that act as nodal points for convergence of afferent and
descending signals as well as the connections between premotor
interneurons in reflex pathways (Jankowska 1992
). Some
of the arrows represent feedback loops between the elements within the
CPG and premotor interneurons, which can adjust rhythm generation to
match external conditions. In addition, all of the segmental elements
project to supraspinal levels, where they enter multiple loops that
project downward to control the segmental circuitry (Armstrong
1988
; Arshavsky et al. 1988
; Drew et al.
1996
).
|
Hultborn and colleagues (1998) have emphasized
the importance of rhythm resetting (persistent phase shift) by afferent
input during fictive locomotion in determining whether a given reflex pathway has direct access to, or may even be part of, the
rhythm-generating network in the locomotor CPG. Usually such resetting
is produced by a short, high-frequency trains of afferent activation,
although Currie and Stein (1988)
demonstrated that
relatively weak cutaneous stimulation can reset ongoing scratching
rhythms in the spinalized turtle. In the present work, we attempted to
avoid major disruptions of ongoing rhythms although, as stated earlier,
we did not attempt to compare locomotor patterns with and without the
low frequency, low amplitude stimuli used to probe reflex pathways. The
following discussion is therefore confined to states of the CPG during
the delivery of such probing stimuli.
Possible wiring diagram
The observations presented in this paper indicate that the
locomotor CPG modulates oligosynaptic excitation of FDL motoneurons in
different ways during fictive locomotion. The provisional wiring diagram in Fig. 12 attempts to tie
together the various findings. This diagram should be considered as an
extension of Fig. 11 because it omits the afferent and descending
pathways that can influence rhythm generation and pattern formation.
The CPG for locomotion (or any other coordinated, rhythmic movement)
has two essential components: an oscillator to generate the basic
rhythm and a system to shape that rhythm into a spatiotemporal pattern
of signals to be delivered to the effector elements (Lennard and
Hermanson 1985; Perret and Cabelguen 1980
). In
some invertebrate CPGs, these two functions are embodied in the same
neural elements. For example, the network that generates, shapes, and
executes the rhythms produced in the stomatogastric ganglion of
crustacea is composed mostly of motoneurons (Selverston et al.
1998
). The structure of the locomotor CPG in mammals remains
unknown, although a variety of conceptual models have been advanced
(reviewed in Grillner 1981
; see also Stein and
Smith 1997
). Although it may be that some neural elements in
the mammalian CPG contribute to both rhythm generation and pattern
formation, we argue in the following text that the two functions are
embodied, at least to some extent, in distinct neural circuits in the
cat.
|
The diagram in Fig. 12 implies that the primary mechanism that produces
modulation in reflex pathway EPSPs is convergence of afferent input and
CPG drive onto common interneurons (Lundberg 1975).
There is clear evidence that primary afferent depolarization (PAD),
which is often associated with presynaptic inhibition, exhibits phasic
variations during fictive locomotion (reviewed in Rossignol
1996
). Although we cannot rule out some participation of
presynaptic inhibition in the observed EPSP modulations, the evidence
available suggests that premotoneuronal convergence onto common
interneurons is the major mechanism responsible (Degtyarenko et
al. 1998b
; Moschovakis et al. 1991
). We also
cannot rule out the possibility that disinhibition could produce some
of the excitatory effects implied by the arrows in the Fig. 12.
Rhythm generation
For the present purpose, we envision the rhythm generation
function as embodied in two reciprocally organized half-centers represented by the split yin-yang symbol in Figs. 11 and 12. In agreement with Hultborn and colleagues (1998), we use
the term "half-center" only as a convenient shorthand, without
implying any specific neural circuitry. The flexor and extensor
half-centers are usually tightly interlocked (heavy arrows), but under
unusual conditions (e.g., Figs. 9 and 10), they can cycle with relative independence (Fig. 12, thin dashed arrows). The alternating half-center outputs are delivered to the ultimate targets (last-order interneurons that excite or inhibit motoneurons) through a system of neurons that
produce the required spatiotemporal sequence of commands. Some of the
last-order target interneurons may be specialized to produce
postsynaptic locomotor drive potentials in motoneurons (LDP INs
in Fig. 12), while others that belong to a variety of reflex pathways
may or may not also contribute to LDPs (see following text).
Pattern formation
The existence of a pattern forming network separate from rhythm
generation is suggested by several pieces of evidence. For example, the
usual firing of FDL motoneurons, accompanied by depolarizing LDPs, can
sometimes be suppressed by varying background afferent input with
little change in the stepping rhythm (Figs. 3 and 4). More
dramatically, FDL can sometimes be made to fire in either F1 or during
extension by changing the steady afferent background, although this
switch is often accompanied by changes in cycle durations (Fig. 6) (see
also Degtyarenko et al. 1998a, their Fig. 8;
Fleshman et al. 1984
, their Figs. 7-11;
Moschovakis et al. 1991
, their Fig. 10). The fact that
F1 facilitation of SP EPSPs in FDL motoneurons is observed whether or
not the FDL motor pool is active in F1 or during extension (Figs. 5 and
6) seems best explained by postulating alternative circuits that are
driven by, rather than integral to, the neural mechanism that produces the overall rhythm. Third, the apparent independent cycling of extensor
(Fig. 9) and flexor half-centers (Fig. 10), with irregular emission of
organized flexion or extension motoneuron bursts and associated
modulation of reflex pathway interneurons, suggests that the
interjected bursts are organized by circuits external to the rhythm
generator and are triggered by it.
Finally, the available evidence indicates a rather precise sequencing
of drive during the flexion phase that is delivered to FDL and other
motoneurons as well as to last-order interneurons that project to them
(Figs. 3A, 6A, 7, and 9A) (see
also Burke 1999; Degtyarenko et al. 1996
,
1998a
,b
; Moschovakis et al. 1991
). The uniformly
brief flexion bursts shown in Fig. 9 give the appearance of stereotyped
event sequences that are triggered with variable phase relations to the
LGS cycling rather than being shaped by the overall rhythm. In the
usual form of fictive locomotion, FDL motoneurons are depolarized and
fire only during the first third of the flexion phase (F1), and they
are actively inhibited during the following two-thirds (F2 and F3;
Figs. 3A, 4C, 6A, 7, and 9A). The F1 phase is also characterized by facilitation of
transmission through the disynaptic excitatory SP pathway to FDL
motoneurons (Figs. 4, 5, 6, 7, and 9) as well as facilitation of the
disynaptic inhibitory pathway to EDL cells (Degtyarenko et al.
1996
). The odd stepping pattern shown in Fig. 7 provides
additional evidence that the pattern forming system that operates
during F1 differ from that driving events later in flexion. We
therefore suggest that the F1
F2
F3 sequencing (denoted by the
arrow linking the F1 and F2 and F3 portions in Fig. 12) is controlled
by mechanisms external to the rhythm generator. Although we cannot rule
out some form of "ring" organization within the rhythm generation circuits themselves (Shik and Orlovsky 1976
), the
existence of independent cycling of flexor and extensor half-centers
(Figs. 9 and 10) is difficult to reconcile with this idea.
Distribution of coordinated drive to motoneurons and interneurons
Locomotor drive from the pattern formation networks must eventually arrive at the appropriate targets at the correct moment during the step cycle. The fact that FDL motoneurons can fire during F1 or during extension, without disrupting the F2-F3 inhibition of these cells or the facilitation of the SP EPSP and the suppression of oligosynaptic MPL EPSPs (Figs. 5 and 6), suggests that the existence of neural circuits intermediate between the basic sequencing layer, represented by the boxes in Fig. 12, and the target cells. The facultative operation of the pattern forming network is embodied in the switch included in the drive pathways between the F1 and Extension elements and the last-order excitatory LDP interneurons that drive FDL motoneurons.
Repetitive stimulation of low-threshold SP afferents during fictive
stepping tends to favor the F1 firing behavior (Fig. 3) (Fleshman et al. 1984, their Fig. 10). On the other
hand, repetitive MPL stimulation can either suppress this drive (Fig.
3) (see also Degtyarenko et al. 1998a
, their Fig. 8) or,
on some occasions, produce extension phase firing of FDL
(Moschovakis et al. 1991
, their Fig. 10). Stimulation of
the sural nerve can also produce extension phase FDL firing
(Fleshman et al. 1984
, their Fig. 9). These observations
suggest that afferent information has access to, and can modify,
pattern formation, presumably to permit adaptive interactions between
the centrally generated pattern and afferent information. Such
relatively subtle effects of afferent input on pattern are included in
Fig. 12 as thin dashed arrows linking SP and MPL afferents to the left
switch. As noted in the preceding text, the diagram does not include
the important mechanisms for resetting of locomotor rhythms
(Hultborn et al. 1998
; Pearson et al.
1998
; Whelan 1996
), which demonstrate that some
afferent systems also have access to the rhythm generation elements in the locomotor CPG.
Locomotor control of transmission through the pathway from
low-threshold MPL afferents and FDL motoneurons provides another example of state-dependent switching. In most examples of fictive stepping, di- and trisynaptic MPL EPSPs are powerfully suppressed throughout the flexion phase (Figs. 1 and 2), as symbolized in Fig. 12
by arrows from the F1 and F2-F3 boxes to inhibitory interneurons that
project to the disynaptic cells in the MPL pathway. However, under some
conditions, trisynaptic MPL EPSPs can be
facilitated during the extension phase of fictive
locomotion. This facilitation is sometimes linked to extensor phase FDL
firing (Moschovakis et al. 1991, their Figs. 9 and 10),
but this linkage is not always present (see Fig. 6D).
Although there is little evidence in the present material for
stereotypical sequencing of extensor muscle activities and reflex pathway control during the extension phase of fictive stepping in out
material, we include a complementary extension pattern formation
network in the conceptual diagram in Fig. 12 because extensor phase
sequencing clearly occurs in extensor muscles in the intact cat (e.g.,
Carlson-Kuhta et al. 1998). The extensor network
enhances transmission of disynaptic group I EPSPs in FDL motoneurons
(8/8 cells studied by Degtyarenko et al. 1998b
; see also
Angel et al. 1996
, their Fig. 5), as it does in other
cat hindlimb muscles, both flexor and extensor (Angel et al.
1996
; Degtyarenko et al. 1998b
; McCrea et
al. 1995
). Disynaptic EPSPs produced in FDL motoneurons by
stimulation of the medial longitudinal fasciculus (MLF) in the brain
stem are also facilitated exclusively during the extension phase,
regardless of the phasing of FDL activity (not included in Fig. 12)
(Degtyarenko et al. 1998a
).
Are last-order interneurons part of the locomotor CPG?
With currently available information, it is impossible to define neurons that are "part" of the locomotor CPG versus neurons that are driven by it, particularly if some of the driven neurons have feedback access to the CPG per se (Fig. 11). Nevertheless, it seems logical to consider that last-order interneurons in reflex pathways, like motoneurons, are targets of CPG control rather than active participants in the rhythm and pattern formation networks. Some of these interneurons could contribute to LDPs when they receive excitatory drive coincident with depolarizing LDPs in the motoneurons, as is the case when disynaptic SP EPSPs are enhanced when FDL fires in F1. However, F1 enhancement of transmission in the SP pathway is sometimes dissociated from F1 depolarization in FDL motoneurons (Figs. 4 and 5), which requires us to postulate the existence of a separate group of excitatory LDP interneurons, as in Fig. 12. The interneurons that produced the oligosynaptic SP EPSPs in the LGS motoneuron shown in Fig. 10 could not have participated in the observed depolarizing LDPs in the cell, because the EPSPs were up-modulated out of phase with those LDPs.
On the other hand, stimulation of the MLR generates EPSPs with
disynaptic segmental latencies in many types of motoneurons (Degtyarenko et al. 1998a; Shefchyk and Jordan
1985
). These disynaptic MLR-evoked EPSP are generally enhanced
during the phase of locomotion in which the recorded motoneurons is
depolarized and active. This is also true of MLR EPSPs in FDL
motoneurons, which are facilitated appropriately whether FDL is active
in F1 or during extension (Degtyarenko et al. 1998a
,
their Fig. 8). This evidence is consistent with the idea that at least
some of the last-order interneurons that generate depolarizing LDPs in
FDL cells also receive direct descending input from the MLR (Fig. 12)
(Jordan 1991
; Shefchyk and Jordan 1985
).
It seems unlikely, however, that all such interneurons are "part"
of the pattern formation function of the locomotor CPG, because the
switching of depolarizing LDP drive to FDL cells appears to be
accomplished at a relatively late stage of pattern formation (Fig. 12).
Structure of the locomotor CPG
The diagram in Fig. 12 is certainly overly simplified even for a
scope limited to systems that project to the FDL motor pool. It is
offered primarily as a framework for discussion of a potentially confusing array of observations and not as a comprehensive model of the
CPG for locomotion in the cat spinal cord. The observations in this
paper do not constrain models of rhythm generation that have been
proposed by others (Grillner 1981; Jankowska et
al. 1967
; Lundberg 1975
; Pearson
1981
; Shik and Orlovsky 1976
), except to suggest
that the half-centers can at times operate semi-autonomously. Figure 12
does not include the cooperative interactions between the circuits that
drive other segmental motor pools and other spinal segments, including
the other limbs, or the influence of brain stem and cerebellum, which
operate in the decerebrate preparation to produce output patterns that
more closely resemble normal walking than those emitted by the isolated
spinal cord (Armstrong 1988
; Grillner
1981
). It also does not include feedback from the pattern forming network to the rhythm-generating system (Fig. 11), which must
be present to adapt the base frequency to the durations of the phased
commands issued to target neurons.
On the other hand, the suggested schematic has the capacity to produce
alternative patterns, embodied in the two switches, that are required
to fit the two firing regimes exhibited by the FDL motor pool. Indeed,
this versatility is the reason for our concentration on this motor
nucleus, and it is also the feature that seems to require the kind of
hierarchical CPG organization suggested in Fig. 12. In his seminal 1981 review on locomotion, Grillner (1981) discussed evidence
that the spinal CPG for locomotion in mammals exhibits such a wide
range of adaptive variability that it cannot be considered a
"hard-wired" system. In recent years, the same plasticity has
become evident even for the best-studied invertebrate CPGs (e.g.,
Harris-Warrick et al. 1998
; Katz 1998
; Selverston et al. 1998
). Adaptability and alternative
usage of shared circuits in the mammalian locomotor CPG, sometimes
termed "modular organization" (Stein and Smith 1997
;
see also Getting 1989
; Jordan 1991
), is
clearly implied in the present results and is therefore embodied in
Fig. 12.
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ACKNOWLEDGMENTS |
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Present addresses: A. M. Degtyarenko, Internal Medicine/Cardiology, TB 172/Campus Bioletti Way, University of California, Davis, CA 95616; E. S. Simon, Movement Disorders Unit, Dept. of Neurology, Tel-Aviv Medical Center, 6 Weizmann St., Tel-Aviv 64239, Israel.
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FOOTNOTES |
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Address for reprint requests: R. E. Burke, Lab. of Neural Control, NINDS, Bldg. 49, Rm. 3A50, National Institutes of Health, Bethesda, MD 20892-4455 (E-mail: reburke{at}helix.nih.gov).
Received 25 October 2000; accepted in final form 20 February 2001.
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REFERENCES |
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