Department of Neuroscience, University of California, Riverside, California 92521
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ABSTRACT |
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Currie, Scott N. and Gregory G. Gonsalves. Reciprocal interactions in the turtle hindlimb enlargement contribute to scratch rhythmogenesis. We examined interactions between the spinal networks that generate right and left rostral scratch motor patterns in turtle hindlimb motoneurons before and after transecting the spinal cord within the anterior hindlimb enlargement. Our results provide evidence that reciprocal inhibition between hip circuit modules can generate hip rhythmicity during the rostral scratch reflex. "Module" refers here to the group of coactive motoneurons and interneurons that controls either flexion or extension of the hip on one side and coordinates that activity with synergist and antagonist motor pools in the same limb and in the contralateral limb. The "bilateral shared core" hypothesis states that hip flexor and extensor (HF and HE) circuit modules interact via crossed and uncrossed spinal pathways: HF modules make reciprocal inhibitory connections with contralateral HF and ipsilateral HE modules and mutual excitatory connections with contralateral HE modules. It is currently unclear how much reciprocal inhibition between modules contributes to scratch rhythmogenesis. To address this issue, fictive scratch motor patterns were recorded bilaterally as electroneurograms from HF, HE, knee extensor (KE), and respiratory (d.D8) muscle nerves in immobilized animals. D3-end (low-spinal) preparations had intact spinal cords posterior to a complete D2-D3 transection. Unilateral stimulation of rostral scratch in D3-end turtles elicited rhythmic alternation between ipsilateral HF and HE bursts in most cycles; consecutive HF bursts were separated by complete silent (HF-OFF ) periods. D3-D9 and D3-D8 preparations received a second spinal transection at the caudal end of segment D9 or D8, respectively, within the anterior hindlimb enlargement. This second transection disconnected most HE circuitry (located mainly in segments D10-S2 of the posterior enlargement) from the rostral scratch network and thereby reduced the HE-associated inhibition of HF circuitry. Unilateral stimulation of rostral scratch in most D3-D9 and D3-D8 preparations evoked rhythmic or weakly modulated ipsilateral HF discharge without HF-OFF periods between bursts and without ipsilateral HE activity in the majority of cycles. In contrast, bilateral stimulation in D3-D9 and D3-D8 preparations reconstructed the HF-OFF periods, increased HF rhythmicity (assessed by fast Fourier transform power spectra and autocorrelation analyses), and reestablished weak HE-phase motoneuron activity. We suggest that bilateral stimulation produced these effects by simultaneously activating reciprocally inhibitory hip modules on opposite sides (right and left HF) and the same side (HF and residual ipsilateral HE circuitry). Our data support the hypothesis that reciprocal inhibition can contribute to spinal rhythmogenesis during the scratch reflex.
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INTRODUCTION |
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A low-spinal turtle that is immobilized by
neuromuscular blockade responds to cutaneous stimulation of the lateral
midbody by generating a fictive rostral scratch motor pattern in
ipsilateral hindlimb motoneurons (Robertson et al.
1985). Rostral cutaneous sensory input enters the midbody
spinal cord segments (D3-D6) and activates
rhythmic motor output from the hindlimb enlargement (D8-D10, S1-S2:
innervating hindlimb muscles) and prehindlimb-enlargement segments
(D6-D7: innervating respiratory muscles). In
D3-end preparations (low spinal animals with a complete
cord transection between segments D2 and D3),
unilaterally evoked fictive rostral scratching is characterized by
rhythmic alternation between ipsilateral hip flexor (HF) and hip
extensor (HE) bursts and monoarticular knee extensor (KE) discharge
during the late HF phase (Robertson et al. 1985
). There
is also weaker rhythmic motor output from contralateral hindlimb
(Currie and Lee 1997
; Stein et al. 1995
,
1998
) and preenlargement (Currie and Gonsalves
1997
) muscle nerves that alternates with ipsilateral activity.
Simultaneous bilateral stimulation in the right and left rostral
receptive fields elicits bilateral rostral scratch motor patterns in
which homologous nerve activity alternates on the right and left sides
(Currie and Gonsalves 1997
; Currie and Lee
1997
; Stein et al. 1995
, 1998
). These results
establish that there is an alternating phase relationship, probably
involving reciprocal inhibition, between mirror-image hip circuitry
(e.g., HF) on the right and left sides and between hip antagonist
circuitry (HF and HE) on the same side during fictive rostral
scratching. Recent experiments in our laboratory indicated that
reciprocal inhibition could generate rhythmic activation of respiratory
motoneurons in the prehindlimb-enlargement turtle spinal cord
(Currie and Gonsalves 1997
). In the present experiments,
we assessed the ability of reciprocal inhibition between hip
circuit modules to generate rhythmic motoneuron activity. According to
the "bilateral shared core" model (Stein et al.
1995
), HF and HE modules, comprising the core of the scratch
rhythm generator, interact via crossed and uncrossed synaptic pathways:
HF modules make reciprocal inhibitory connections with contralateral HF
and ipsilateral HE modules and mutual excitatory connections with
contralateral HE modules. These connections are supported by more
recent studies (Currie 1997
; Currie and Lee
1997
; Stein et al. 1998
). We disconnected as
much HE circuitry as possible from the right and left rostral scratch networks by completely transecting the spinal cord at the caudal end of
segment D9 or D8 in the anterior enlargement;
this created D3-D9 and
D3-D8 preparations, respectively. The cell
bodies of HE motoneurons are distributed from segment D9 to
S2 at the posterior end of the enlargement and are most
concentrated in segments D10-S2 (Ruigrok and Crowe 1984
). HF motoneurons are distributed
more anteriorly, in segments D8-D9. Lesion
experiments by Mortin and Stein (1989)
suggested that at
least some of the premotor circuitry for HF and HE was located in the
same segments as the motoneurons. Those authors created isolated
D8 and D8-D9 preparations by
completely transecting the spinal cord at the anterior and posterior
ends of those segments. Rhythmic HF motor discharge, lacking quiescent (HF-OFF) periods between bursts, still could be elicited in
these animals by tactile stimulation within the pocket scratch
receptive field. That result showed that sufficient interneuronal
circuitry exists within the D8 and D9 segments
to generate rhythmic discharge in HF motoneurons. Similar rhythmic HF
discharge without HF-OFF periods also was exhibited by
D3-D9 and D3-D8
preparations in that study, indicating that HE circuitry, mainly
located posterior to D9, was required for normal inhibition
of HF motoneurons between bursts. Thus by cutting away the posterior
enlargement in the present experiments, we removed a large fraction of
HE circuitry from the rostral scratch network and greatly reduced the
HE-associated inhibition of ipsilateral HF discharge. Our data show
that during unilateral stimulation of rostral scratch, most
D3-D9 and D3-D8 turtles displayed a low percentage of normal HF cycles with complete off periods between bursts, confirming the earlier work by
Mortin and Stein (1989)
. In contrast, bilateral
stimulation of rostral scratch produced vigorous, alternating discharge
in right and left HF nerves with restored HF-OFF periods on
both sides and greatly increased HF rhythmicity. Our experiments
support the hypothesis that reciprocal inhibition in the anterior
hindlimb enlargement can contribute to hindlimb rhythmogenesis during
rostral scratch motor patterns. Portions of this work were published in an extended abstract (Currie and Gonsalves 1998
).
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METHODS |
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Red-eared slider turtles (n = 11),
Trachemys scripta elegans, weighing 470-650 g, were placed
in crushed ice for a minimum of 2 h before surgery to induce
hypothermic anesthesia (Lennard and Stein 1977). Turtles
were maintained partially immersed in crushed ice during all surgical
procedures. The first surgical procedure was complete transection of
the spinal cord just posterior to the forelimb enlargement, between
spinal segments D2 and D3 (D2 = the
second postcervical segment) (Zangerl 1969
).
Surgical procedures
Hindlimb muscle nerves were prepared bilaterally for
electroneurographic (ENG) recording (see Fig.
1; nerves shown only on right side). The
FT-KE and AM-KE nerves innervate triceps femoris pars
femorotibialis and pars ambiens, respectively. VP-HP innervates puboischiofemoralis internus, pars anteroventralis, a hip flexor muscle. HR-KF innervates several bifunctional hip extensor-knee flexor
muscles of the flexor tibialis group. Distal D8 (d.D8) innervates
respiratory and pelvic muscles adjacent to the hindlimb. These nerves
and the muscles they innervate have been described previously
(Mortin and Stein 1989; Robertson et al.
1985
). Motoneurons with axons in the d.D8 nerve exhibit two
phases of activity during a scratch reflex: a HF-correlated burst and a
HE-correlated burst (Currie and Lee 1996b
; Mortin
and Stein 1989
). We used d. 8 as a monitor of HE activity in
some D3-D8 preparations in which the entire
HR-KF (HE) motor pool was removed by the D8-D9
cord transection. Hereafter in the text, FT-KE and AM-KE are referred
to as KE nerves, VP-HP as the HF nerve, and HR-KF as the HE nerve. In
four turtles, we prepared KE, HF, and HE nerves bilaterally
(experiments 1, 9, 10, and 11). In four other
turtles, we prepared KE, HF, and d.D8 nerves bilaterally
(experiments 2-5). In three turtles, we prepared only KE
and HF nerves bilaterally (experiments 6-8). Each nerve was
freed from surrounding tissues, tied with surgical thread near its
muscle insertion, and then cut distal to the tie.
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We exposed spinal cord segments D8-D10 at the anterior side of the hindlimb enlargement by drilling a dorsal midline channel through the carapace and performing a dorsal laminectomy. The turtle hindlimb enlargement consists of three dorsal segments (D8, D9, D10) and two sacral segments (S1, S2; Fig. 1). In five turtles (experiments 1-5), we covered the exposed cord with saline-soaked Gelfoam surgical sponge so that the spinal cord could be transected later at the caudal end of the D8 (n = 4) or D9 (n = 1) segment, during the experiment. In six turtles (experiments 6-11), we transected the cord during the initial dissection at the caudal end of D8 (n = 2) or D9 (n = 4), while the turtle was still on ice. Transections were performed under a dissection microscope with fine iridectomy scissors. The completeness of transections was confirmed visually by lifting one of the cut ends of the spinal cord upward with forceps, viewing the cord in cross-section, and then replacing it in the spinal canal.
ENG recordings and data storage
After surgery was complete, preparations were allowed to warm up to room temperature and then were immobilized with an intramuscular injection of gallamine triethiodide (8 mg/kg body wt; Sigma, St. Louis, MO). The trachea was intubated and artificial respiration used throughout the experiment. The skin was kept moist with turtle saline. Three separate rings of warm dental wax were formed around the holes in the dorsal carapace over the right and left hindlimb nerves and the exposed spinal cord, allowed to cool and harden, and glued in place with cyanoacrylate adhesive. The dissected hindlimb nerves were arrayed for recording by securing their attached threads to the rim of the wax well. The wells surrounding hindlimb nerves were filled with mineral oil; the well surrounding the exposed spinal cord was filled with turtle saline. Bipolar hook electrodes (100 µm diam silver) were used to record from the nerves, electrically insulated by the mineral oil. ENG signals were amplified and filtered (band-pass 100 Hz to 1 kHz), digitized by a PCM video adapter (Vetter; Rebersburg, PA) and stored along with a voice channel and stimulus marker on videotape (band-pass DC, 3.5 kHz). Hard copies of all recordings from each experiment were printed with an eight-channel thermal-array recorder (Astro-Med; West Warwick, RI). Selected sequences also were redigitized off-line at 2 kHz per channel on a PC computer using a Digidata 1200A A-D converter with Axotape 2.0 software (Axon Instruments; Foster City, CA), formatted with Datapac II (Run Technologies; Laguna Hills, CA) and Coreldraw (Corel; Ottawa, Canada) software, and plotted on a laser printer.
Stimulation
We used either mechanical or electrical stimulation of the shell
to evoke fictive rostral scratch reflexes. Scratch episodes always were
separated by rest periods of 2 min. Mechanical stimulation was
applied by gently rubbing a site on the shell with a fire-polished glass probe. In some cases, the probe was mounted on a hand-held force
transducer (Grass Instruments-Astro-Med) to record the force and timing
of the stimulus. Rubs with the glass probe were applied with a force of
0.2-1.4 N and lasted 15-20 s. Electrical stimulation was applied
either unilaterally or bilaterally to sites in the rostral scratch
receptive field via pin electrodes inserted into the shell epidermis
2-3 mm apart (Currie and Stein 1990
). Pulses of 10- to
20-V amplitude and 1-ms duration were delivered in 50-pulse trains with
an interpulse interval of 320 ms. For experiments in which bilateral
electrical stimulation was applied, pin electrodes were inserted into
mirror-image sites (SP2 or SP2.5) (see Mortin and Stein
1990
) in the right and left rostral scratch receptive fields;
during bilateral stimulation, identical trains of synchronized pulses
were delivered to both sides. Unilateral and bilateral stimulus trains
were applied in the following sequence: right, left, bilateral,
bilateral, right, left (Stein et al. 1995
).
Data analysis
PERCENTAGE OF SCRATCH CYCLES WITH NORMAL HF-OFF PERIODS. To determine the average percentage of rostral (Table 1) or pocket (RESULTS) scratch cycles per episode with normal HF-OFF periods, we examined thermal-array printouts of scratch ENGs. The total number of complete scratch cycles that occurred during stimulation was determined for each episode. Within an episode, we counted the number of "normal" cycles in which the HF ENG burst was followed by a period of complete quiescence (an HF-OFF period; e.g., Fig. 2, A1 and C1) and the number of cycles in which the HF burst was followed by a period of reduced ENG amplitude but not complete quiescence (no HF-OFF period; e.g., Fig. 3, A1 and C1). We defined an HF burst as a period of increased HF ENG amplitude that was accompanied by coactive discharge in the ipsilateral KE (AM-KE or FT-KE for rostral scratch episodes; AM-KE for pocket scratch episodes) or the contralateral HE nerve. In three D3-D8 preparations (experiments 2, 4, and 5), clear HF bursts could not be reliably identified during unilateral stimulation of rostral scratch (e.g., Fig. 3A2); these experiments were excluded from the analysis in Table 1.
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RECTIFIED-SMOOTHED ENG RECORDINGS. Rectified-smoothed ENG data (shown in Figs. 4, A and B, and 5 and used in time series analyses) were prepared by digitizing HF nerve recordings (100 Hz to 1 kHz bandwidth) off-line at 2 kHz per channel; digitized files then were full-wave rectified, rebinned at 100 Hz with a 100-point nonmoving average (50-ms binwidth), and smoothed with an even-weighted 10-point moving average (100-ms binwidth), using Datapac II software.
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TIME SERIES ANALYSES. Time series analyses were used to examine rectified-smoothed HF nerve recordings for periodic trends in burst amplitude (autocorrelation, power spectra) and to identify relations between HF bursts on the right and left sides and any time delays between those bursts (cross-correlation). For autocorrelations, recordings from the left HF nerve, each consisting of 1,024 data points (10.23 s at a 100-Hz sampling rate) and beginning 3 s after stimulus-onset, were obtained from four consecutive rostral scratch episodes evoked by left stimulation (Fig. 4C) and four consecutive episodes evoked by bilateral stimulation (Fig. 4D) in experiment 2. Left-stimulation data were concatenated end to end into a single 4,096-point ASCII file; bilateral-stimulation data were concatenated into a second ASCII file. Both files then were imported into the Systat 7.0 software package (SPSS; Chicago, IL) for analysis. Cross-correlation (Fig. 5B) was performed on the same four bilateral-stimulation episodes used in Fig. 4D by importing both left and right HF recordings (1,024 points per channel), as a single ASCII file, into the Systat program. Plots show the correlation of the time series variable (HF voltage) with itself (autocorrelation) or with the contralateral nerve series (cross-correlation), negatively shifted by a number of time lags = 1, 2, 3, etc. The maximum number of lags in correlation plots was 500, lag duration was 10 ms.
Power spectrum analyses were performed on rectified-smoothed left HF nerve signals with a fast Fourier transform (FFT) program in the Datapac II software package. In Fig. 4, E and F, the average power spectra were calculated for four consecutive rostral scratch episodes evoked by left stimulation (Fig. 4E) and four consecutive episodes evoked by bilateral stimulation (Fig. 4F) in experiment 2, using the same 1,024-point data sequences that were analyzed by autocorrelation in Fig. 4, C and D. The DC offset was removed from each data sequence by calculating the mean voltage for the entire sequence, then subtracting that value from each data point. Removing DC offset greatly reduced low-frequency components (0.0-0.1 Hz) in the power spectra, which when present, tended to mask nearby higher-frequency peaks. In Fig. 6, we calculated the average total power within the 0.29- to 0.49-Hz bandwidth for 16 scratch episodes (4 episodes each from 4 different experiments) in each of the following categories: left stimulation in D3-D8 preparations, bilateral stimulation in D3-D8 preparations, left stimulation in D3-D9 preparations, bilateral stimulation in D3-D9 preparations. The D3-D8 preparations chosen for analysis were experiments 2, 4, 5, and 7; two other experiments (3 and 11) were excluded because they exhibited normal HF-OFF periods in >50% of rostral scratch cycles during left stimulation (see Table 1). The D3-D9 preparations were experiments 1, 6, 9, and 10; one other experiment (8) was excluded because of an insufficient number of scratch episodes. We calculated total power as
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RESULTS |
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Unilateral and bilateral stimulation of rostral scratch in D3-end preparations
UNILATERAL STIMULATION.
Bilateral ENG recordings were obtained from hindlimb (HF, HE, KE) and
respiratory (d.D8) muscle nerves during fictive rostral scratch motor
patterns in five D3-end preparations that had intact spinal
cords posterior to a D2-D3 transection site
(Fig. 1). Fictive rostral scratching was elicited by mechanical or
electrical stimulation (see METHODS) of the shell-surface
within the rostral receptive field, located on the lateral
"shell-bridge," anterior to the hindlimb (Mortin and Stein
1990; Mortin et al. 1985
). In one
D3-end preparation, we recorded from KE, HF, and HE nerves
bilaterally (Fig. 2, A1-C1). During unilateral stimulation
in the rostral receptive field, this turtle exhibited normal rhythmic
alternation between HF and HE bursts ipsilateral to the stimulus and KE
discharge during the late HF phase of each scratch cycle (Fig. 2,
A1 and C1) (see also Robertson et al.
1985
). There was also weak rhythmic discharge in contralateral
HE (Fig. 2, A1 and C1) and HF (Fig. 2A1) nerves that was out-of-phase with ipsilateral activity
(see also Currie and Lee 1997
; Stein et al.
1995
). In four other D3-end preparations, we
recorded from KE, HF, and d.D8 nerves bilaterally (e.g., Fig. 2,
A2-C2), using d.D8 as a monitor of the HE phase of the
scratch. Motoneurons with axons in the d.D8 nerve innervate respiratory
muscle and exhibit two phases of activity during a scratch reflex: an
HF-correlated burst and an HE-correlated burst (Currie and Lee
1996b
; Mortin and Stein 1989
). Among all
D3-end preparations, the average percentage of rostral
scratch cycles per episode with normal HF-off periods was 86.6 ± 18.6% (62 episodes in 5 turtles) during unilateral stimulation. This
is very close to the percentage of normal cycles observed during
unilateral stimulation in previous work with D3-end
preparations (84.1%) (see Stein et al. 1998
).
BILATERAL STIMULATION.
Simultaneous stimulation of mirror-image sites in the right and left
rostral receptive fields evoked bilateral rostral scratching in which
the activity of homologous nerves (e.g., HF) alternated on the right
and left sides (Fig. 2B, 1 and 2) (see also
Currie and Lee 1997; Stein et al. 1995
,
1998
). We observed right-left alternation of HF discharge
during bilateral rostral scratching in all five D3-end
preparations in this study. We include these examples of normal
unilateral and bilateral rostral scratching in the present paper so
that direct comparisons can be made with the responses of
D3-D9 and D3-D8
preparations, in which most HE circuitry was disconnected (see Fig. 3,
following text).
Unilateral and bilateral stimulation of rostral scratch in D3-D9 and D3-D8 preparations
The D3-D9 and
D3-D8 preparations in this study were created
by a second spinal transection at the caudal end of the D9
or D8 segment, respectively, within the anterior hindlimb
enlargement (Fig. 1). The somata of most HE motoneurons reside
posterior to these transections in segments
D10-S2, whereas HF motoneurons are distributed
in segments D8-D9 (Ruigrok and Crowe
1984). Spinal transection experiments performed by
Mortin and Stein (1989)
indicated that some of the
interneuronal circuitry associated with the HF and HE modules was
located in the same segments as their respective motoneurons. Thus it
is likely that our transections in the anterior enlargement
disconnected most HE motoneurons and some associated premotor circuitry
from the right and left rostral scratch networks. In five turtles, we
transected at the caudal end of D9 (n = 1) or D8 (n = 4) after first recording
D3-end responses (see preceding text). In six other
turtles, we transected the cord at the caudal end of D9
(n = 4) or D8 (n = 2)
during the initial dissection, without recording D3-end
responses. All preparations were allowed
20 min to recover from cord
transection before recording scratch responses. Turtles did not exhibit
significant spinal shock because shell stimulation evoked robust
fictive scratching within a few minutes after spinal transection (see
also Mortin and Stein 1989
). No significant change was
noted in the rhythmicity or percentage of HF-OFF periods
when comparing scratch responses 20-30 min after transection with
responses obtained >12 h after transection (data not shown).
Because our transections in the enlargement removed most or all HE
motoneurons in the majority of preparations, we did not always monitor
HE motor output (see Surgical procedures). We focused instead on HF activity during unilateral and bilateral scratching because a large fraction of the HF motor pool and associated premotor circuitry is located within the D8 segment (Mortin
and Stein 1989; Ruigrok and Crowe 1984
) and so,
remained connected to the rostral scratch network in all
D3-D9 and D3-D8
preparations. Some of our unilaterally evoked rostral scratch episodes
(e.g., Fig. 3, A1 and C1) contained cycles that
could be described as "HE deletions." HE deletions were defined
in previous work as rostral scratch cycles that exhibit consecutive HF
bursts without clear quiescent (HF-OFF) periods separating
them and without corresponding HE bursts; to define these as rostral
scratch cycles, the monoarticular knee extensor FT-KE also must be
active during the latter part of each HF burst (Stein et al.
1995
). In many of our D3-D9 and D3-D8 responses, HE activity was not recorded
(e.g., Fig. 3, A2-C2), and the FT-KE nerve was often
inactive during some cycles (e.g., latter part of Fig. 3C1)
or during the entire scratch episode (Fig. 3, A2-C2). It
was not possible to define such cycles as HE deletions, therefore we
avoided that terminology. We refer instead to HF cycles with or without
HF-OFF periods (see Table 1).
PERCENTAGE OF CYCLES WITH HF-OFF PERIODS DURING
UNILATERAL AND BILATERAL STIMULATION.
The D3-D9 preparation in Fig. 3,
A1-C1, displayed strikingly different motor output within a
given hindlimb in response to unilateral and bilateral stimulation of
rostral scratch receptive fields. Unilaterally evoked responses did not
exhibit normal alternation between HF and HE bursts during the period
of stimulation (Fig. 3, A1 and C1). Instead,
these episodes displayed continuous, rhythmically modulated output from
the ipsilateral HF nerve and weak KE discharge near the end of some,
but not all HF bursts; there were no quiescent (HF-OFF)
periods between HF bursts and no associated ipsilateral HE discharge
during the period of stimulation. It is interesting, however, that both
of the unilaterally evoked responses displayed a cycle of normal HF-HE
alternation, with a complete HF-OFF period, after the end
of the stimulus train. Such "HE afterdischarges" were very common
during unilaterally evoked rostral scratch episodes in all three
D3-D9 preparations in which we recorded from
KE, HF, and HE nerves; we also observed the HF-OFF period
after stimulus-offset in two other D3-D9
preparations where we recorded only KE and HF nerves. In contrast to
unilaterally elicited responses, bilateral stimulation of rostral
receptive fields reconstructed normal rostral scratch cycles with
complete HF-OFF periods, near-normal HE burst-amplitudes, and strong KE discharge during the period of stimulation (Fig. 3B1). This result is consistent with the hypothesis of
Stein et al. (1995; their Fig. 9) that HF modules make
reciprocal inhibitory connections with contralateral HF and ipsilateral
HE circuitry and mutual excitatory connections with contralateral HE circuitry.
COMPARISON OF HF RHYTHMICITY DURING UNILATERAL AND BILATERAL
STIMULATION.
In three of six D3-D8 turtles, unilateral
stimulation of the rostral receptive field elicited only irregular,
modulated discharge in the ipsilateral HF and d.D8 nerves; individual
HF bursts could not be reliably identified during unilateral
stimulation (e.g., Fig. 3A2). In these preparations,
bilateral stimulation did not reestablish complete HF-OFF
periods but did significantly increase HF "rhythmicity" (regular
periodic modulations of ENG amplitude) compared with unilateral
stimulation (e.g., Fig. 3B2). During bilaterally evoked
responses, distinct bursting appeared de novo in the right and left HF
recordings and alternated from side to side. Small HE phase bursts also
were displayed by the right d.D8 respiratory nerve during the low
points between right HF bursts (Fig. 3B2, ), indicating
that some HE-associated circuitry was present within the
D3-D8 spinal cord and could be activated
during bilateral stimulation. The increase in HF rhythmicity that
occurred during bilateral stimulation was even more apparent in
rectified-smoothed ENG recordings (Fig. 4, A and
B) and in autocorrelation plots (Fig. 4, C and
D). Spectral analyses were also performed using an FFT
algorithm (Fig. 4, E and F); these showed that
during bilateral stimulation, a large power peak developed at a
frequency of 0.39 Hz, corresponding to an average cycle period of
2.56 s. This value is in good agreement with the average cycle
period measured between the first and second positive peaks in the
autocorrelogram (2.67 s; Fig. 4D).
Unilateral stimulation of pocket scratch in D3-D9 and D3-D8 preparations
Fictive pocket scratching was elicited by mechanical stimulation
of the shell-skin border within the ventral pocket receptive field,
located ventral and anterior to the thigh within the "hindlimb pocket" region (Mortin and Stein 1990; Mortin
et al. 1985
). Normal rostral and pocket scratch motor patterns
in D3-end preparations exhibit different knee extensor
(AM-KE and FT-KE) timing within the HF-HE activity cycle
(Robertson et al. 1985
). In the rostral scratch, both
FT-KE and AM-KE bursts begin and end during the HF phase. During the
pocket scratch, FT-KE bursts begin and end during the HE
(HF-OFF) phase, whereas AM-KE bursts begin during the HF
phase and cease during the HE phase. In the present experiments, we
compared HF activity during fictive pocket and rostral scratch responses in D3-D9 and
D3-D8 turtles. Unilaterally evoked pocket scratch responses displayed a high percentage of normal HF cycles with
clear HF-OFF periods in D3-D9 and
D3-D8 preparations, in contrast to the
near-zero percentage observed during unilaterally evoked rostral
scratching in the same preparations. Figure
7A shows a rostral scratch
episode from a D3-D9 preparation in which there was rhythmic HF discharge with no HF-OFF periods
between most bursts. Figure 7B shows a pocket scratch
episode from this same animal in which sharply defined HF bursts
exhibited abrupt terminations and distinct OFF periods in
every cycle. In two turtles, we had a sufficient number of pocket
scratch episodes to permit a statistical comparison with rostral
scratch episodes. The average percentage of cycles per episode with
complete HF-OFF periods during unilateral rostral scratch
and unilateral pocket scratch stimulation were as follows (average ± SD): experiment 7, a D3-D8 preparation (rostral, 10 episodes): 3.3 ± 7.0%, (pocket, 10 episodes): 91.4 ± 5.3%; experiment 9, a
D3-D9 preparation (rostral, 12 episodes): 15.6 ± 18.8%, (pocket, 7 episodes): 100.0 ± 0.0%. In both
experiments, the pocket scratch average was significantly higher than
the rostral scratch average at P < 0.0002, using the
Mann-Whitney U test (Siegel 1956
). One
interpretation of these data is that some interneuronal circuitry that
inhibits HF is present within the D8 and D9
segments but is not sufficiently activated by unilateral rostral
stimulation to completely terminate HF bursts. Pocket stimulation may
provide stronger drive to these inhibitory elements, resulting in more complete HF burst terminations. This view also is supported by previous
work that demonstrated normal pocket scratch cycles in surgically
restricted spinal cord preparations (Currie and Lee 1996b
; Mortin and Stein 1989
).
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DISCUSSION |
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The goal of the present experiments was to investigate mechanisms
of hip rhythmogenesis during fictive rostral scratching in turtles. Our
results provide evidence that reciprocal inhibition between hip circuit
modules in the anterior hindlimb enlargement can generate rhythmicity
during bilateral hindlimb motor patterns. We use the term
"module" to describe the group of coactive neuronal elements that
control the activity of agonist (e.g., HF) motoneurons for one limb and
coordinate the activity with that of synergist and antagonist motor
pools in the same limb and in the contralateral limb (Jordan
1991; Jordan et al. 1986
; Stein and Smith
1997
; Stein et al. 1995
). We attach special
significance to hip rhythm generation because previous work that
examined phase-dependent resets demonstrated that hip (but not knee)
motor output is linked tightly to the timing of the fictive scratch
rhythm for the entire limb (Currie and Stein 1989
). The
importance of hip circuitry in the control of the overall limb rhythm
also was demonstrated by Andersson and Grillner (1981
,
1983
) for fictive stepping in cats. We focus on HF activity in
part because HF motoneurons (Ruigrok and Crowe 1984
) and
at least some HF premotor circuitry (Mortin and Stein 1989
) are located in the anterior two segments of the spinal
hindlimb enlargement (D8 and D9), while most HE
circuitry is located in more posterior segments (D10,
S1, S2). The anterior segments of hindlimb
enlargement are known to display the greatest rhythmogenic capacity for
turtle (Mortin and Stein 1989
) and cat (Deliagina et al. 1983
; Gelfand et al. 1988
) scratching as
well as for locomotor activity in various preparations (neonatal rat:
Cowley and Schmidt 1997
; Kjaerulff and Kiehn
1996
; cat: Grillner and Zangger 1979
; chick
embryo: Ho and O'Donovan 1993
).
The bilateral shared core model of the turtle rostral scratch CPG
proposes that HF and HE modules make reciprocal inhibitory connections
with each other on the same side (e.g., right HF-HE), and with their
mirror-image homologs on the opposite side (e.g., right-left HF)
(Stein et al. 1995). In addition, the model suggests that HF modules make mutual crossed excitatory connections with contralateral HE modules. It is currently unknown to what extent individual HF and HE modules can be independently rhythmogenic and
whether rhythmicity can be produced in the scratch network by
reciprocal interactions between modules. The goal of the present experiments was to assess the capability of reciprocal inhibition between hip modules to contribute to scratch rhythmogenesis. Our approach was to transect the spinal cord at the posterior end of
segment D9 or D8 in the anterior hindlimb
enlargement, which disconnected as much HE circuitry as possible from
the right and left rostral scratch networks (Figs. 1 and
8, A1-C1). Most of the
resulting D3-D9 and
D3-D8 preparations responded to unilateral stimulation of the rostral scratch receptive field with rhythmic ipsilateral HF discharge that lacked "HF-OFF" periods
between bursts in most cycles and weak contralateral HE bursts in some preparations that were coactive with ipsilateral HF bursts (Figs. 3,
A1 and C1, and 8, A1, A2, C1, and
C2). Thus unilateral rostral stimulation in
D3-D9 and D3-D8
preparations produced activation (at the level of motoneuron
discharge) of the mutually excitatory ipsilateral HF and contralateral
HE modules alone, generating only weak rhythmicity. In contrast,
bilateral stimulation of rostral scratch, which simultaneously excited
reciprocally inhibitory hip modules on opposite sides (right and left
HF) and on the same side (HF and residual HE circuitry spared by the
transection), evoked vigorous alternating discharge in right and left
HF nerves that exhibited enhanced HF rhythmicity on both sides (Figs.
4, 6, and 8B, 1 and 2) and reconstructed
HF-OFF periods between bursts (Figs. 3 and 8B,
1 and 2, and Table 1). We interpret these results to mean that reciprocal inhibition between hip modules can help to
terminate HF bursts and increase HF rhythmicity under conditions where
mutually inhibitory modules are strongly coactivated (as during
bilateral scratching). Crossed reciprocal inhibition between right and
left HF modules probably contributed the most to these effects,
assuming that relatively little HE circuitry remained in
D3-D9 and D3-D8
preparations to interact with ipsilateral HF modules. Crossed
inhibition also has been proposed to participate in the construction of
axial locomotor rhythms in lampreys, fish, and frog embryos (reviewed
in Fetcho 1991
; Friesen 1994
;
Kiehn et al. 1997
; Marder and Calabrese
1996
). The relative contribution of crossed inhibition to
rhythmogenesis in the turtle hindlimb enlargement can be assessed
further in future experiments by observing the effects of midline
spinal lesions that sever commissural pathways.
|
Evidence from several studies indicates that a single HF module on one
side of the turtle spinal cord may be capable of some independent
rhythmicity in response to rostral scratch sensory input; this is based
on the observation that continuous rhythmic HF discharge (without
ipsilateral HE activity) can occur spontaneously in D3-end
turtles (Robertson and Stein 1988; Robertson et
al. 1985
; Stein and Grossman 1980
; Stein
et al. 1982
) or can be induced to occur with greater frequency
by surgical (Mortin and Stein 1989
; Stein et al.
1995
, 1998
; the present study) or chemical (Currie and
Lee 1997
) manipulations of the spinal cord. Thus these studies
and the present experiments show that a single HF module can express
rhythmic output without activation of the ipsilateral antagonist
motoneurons. Similar independent activation of elbow flexor or extensor
bursting was reported recently for the in vitro mudpuppy spinal
cord (Cheng et al. 1998
). In the present experiments, the turtle spinal cord was able to produce rhythmically modulated HF
discharge on one side without activation of either the ipsilateral antagonist (HE) or the contralateral mirror-image (HF) motoneurons (Fig. 3, A1 and C1). These observations support
the view that some independent HF rhythmicity can be elicited by
scratch stimuli in the absence of either crossed or uncrossed
reciprocal inhibition. Experiments with in vitro mammalian spinal
cords, in which inhibitory neurotransmission was blocked, also have
indicated that limb rhythmogenesis can occur in the absence of synaptic
inhibition (Bracci et al. 1996
; Cazalets et al.
1996
; Cowley and Schmidt 1995
, 1997
; Ho 1997
; Ozaki et al. 1996
). Note, however, that
reciprocal inhibition between hip modules in the turtle need not
necessarily produce motor output from both modules. For example, it
seems plausible that a subset of ipsilateral HE and contralateral HF
interneurons might be active in antiphase with the "independent"
HF bursts on one side, without producing detectable motor output from
ipsilateral HE or contralateral HF. Such "subthreshold"
reciprocal interactions might contribute to the rhythmicity of HF motor
output. Future experiments could begin to address this issue by
searching with an extracellular microelectrode in the ipsilateral HE
(D10-S2) and contralateral HF
(D8-D9) areas of the spinal cord for
interneuronal units that exhibit antiphasic discharge during
independent HF bursting on one side.
It may be argued that bilateral stimulation of rostral scratch could
increase HF rhythmicity and the occurrence of HF-OFF periods not because of reciprocal inhibitory interactions but simply
because it provides more sensory drive to HF modules. A unilateral
rostral stimulus, in transected D3-D9 and
D3-D8 turtles, may not excite the ipsilateral
HF module sufficiently for it to become rhythmically active. We believe
this is unlikely for two reasons. First, one would expect that if HF
modules were more intensely activated by bilateral stimulation, that
this would be reflected in larger HF burst amplitudes. However,
Stein et al. (1995; their Fig. 3A) showed that there was
no significant difference in HF burst amplitudes during unilateral and
bilateral stimulation of rostral scratch in D3-end
preparations. We also did not observe noticeably larger HF bursts
during bilateral rostral stimulation in the present study (e.g., Fig.
2), although we did not quantify burst amplitudes. Second, there is no
basis for believing that more intense activation of HF increases its
rhythmicity and the occurrence of off periods between bursts. In fact,
during a typical rostral scratch episode in D3-end turtles,
spontaneous HE-deletion cycles that lack HF-OFF periods
nearly always occur at the beginning of the episode, when HF burst
amplitudes are greatest (Robertson and Stein 1988
;
Stein et al. 1982
, 1995
).
Rostral and pocket scratch CPGs displayed different capacities to generate normal HF cycles in turtles where most HE circuitry was removed (D3-D9 and D3-D8 preparations). Unilateral rostral stimulation evoked rhythmically modulated discharge in ipsilateral HF motoneurons with a very low percentage of off periods between bursts (Table 1; Figs. 3 and 7A). In contrast, unilateral pocket stimulation elicited vigorous HF bursts separated by distinct, long-lasting off periods in nearly all cycles (Fig. 7B). It is unlikely that the increased occurrence of HF-OFF periods during pocket scratch was due to more intense activation of the HF module by pocket sensory input. Distinct OFF periods were present even between the weakest HF bursts in the pocket scratch (Fig. 7B; bursts 8 and 9) but were absent even between the strongest HF bursts in the rostral scratch (Fig. 7A; bursts 1 and 2). A possible explanation is that some HE-associated interneurons (components of the ipsilateral HE module) that inhibit HF are present within the D8 and D9 segments and are strongly excited by unilateral pocket scratch stimulation but less excited by unilateral rostral scratch stimulation. Activation of these HE-associated inhibitory interneurons may not be sufficient during unilateral rostral stimulation to completely terminate HF bursts. Bilateral rostral stimulation may reconstruct HF-OFF periods and increase HF rhythmicity, in part, by strongly exciting these same HE interneurons (via crossed pathways) that are accessed by unilateral pocket sensory input.
We previously explored the participation of the portion of spinal cord
anterior to the hindlimb enlargement (preenlargement segments
D3-D7) in the generation of turtle rostral
scratch motor rhythms (Currie and Gonsalves 1997). The
D7 spinal cord segment is immediately anterior to the
hindlimb enlargement and contains two populations of motoneurons,
"TD7" and "OD7," that innervate the transverse-abdominus
and oblique-abdominus respiratory muscles, respectively (Currie
and Gonsalves 1997
; Mortin and Stein 1989
). In
D3-end turtles, unilateral stimulation of the right rostral scratch receptive field evoked rhythmic coactivation of right HF, right
TD7, and left OD7 motoneurons that could alternate with weaker coactive
bursts in left TD7 and right OD7. D3-D7
preparations had two complete spinal transections, one at
D2-D3 and a second at
D7-D8 that disconnected the entire hindlimb
enlargement from the rostral scratch network. In
D3-D7 turtles, unilateral stimulation of the
right rostral receptive field evoked only tonic or weakly modulated
discharge in right TD7 and left OD7 respiratory motoneurons. However,
bilateral stimulation of rostral receptive fields reestablished vigorous bursting in which coactive right TD7 and left OD7 bursts alternated with coactive left TD7 and right OD7 bursts. These results
indicated that simultaneous activation of reciprocally inhibitory
circuit modules could generate rhythmicity in preenlargement networks
and therefore implied that similar mechanisms might operate in the
hindlimb enlargement to enhance hip rhythmicity. Furthermore because
the D7 segment exhibited little or no rhythmicity during unilateral stimulation but strong rhythmicity during bilateral stimulation, preenlargement D7 networks may contribute
significantly to the increased rhythmicity that we observed in
D3-D9 and D3-D8 preparations during bilateral stimulation in the present study (Figs.
3, 4, and 6).
Pharmacological experiments support the conclusion that some but not
all inhibition in turtle spinal motor networks is mediated by
strychnine-sensitive glycine receptors (Currie and Lee 1996a, 1997
). During the fictive flexion reflex, turtles exhibited a crossed inhibition of contralateral hip flexor activity (Currie and Lee 1996a
; Currie and Stein 1989
) similar to
that of mammals (Eccles and Sherrington 1931
;
Holmqvist 1961
; Jankowska et al. 1967
;
Sherrington 1906
). The crossed inhibition associated
with flexion reflex appeared to involve glycinergic transmission in turtles because it was abolished and replaced by crossed excitation after strychnine application to the hindlimb enlargement (Currie and Lee 1996a
). However, during bilateral fictive rostral
scratching, prolonged application of 50 µM strychnine to the turtle
hindlimb enlargement produced only a slight increase in the variability of interlimb phase values (right vs. left hip flexor bursts) and did
not abolish right-left (interlimb) or flexor-extensor (intralimb) alternation (Currie and Lee 1997
). That result contrasts
with fictive locomotion studies in which strychnine synchronized the normally alternating discharge in mirror-image right- and left-side muscle nerves (lamprey: Cohen and Harris-Warrick 1984
;
Hagevik and McClellan 1994
; neonatal rat: Cowley
and Schmidt 1995
; Kremer and Lev-Tov 1997
;
Kudo et al. 1991
; cat: Noga et al. 1993
)
and intralimb flexors and extensors (neonatal rat: Cowley and
Schmidt 1995
; cat: Kriellaars et al. 1988
;
Noga et al. 1995
). Bilateral stimulation of rostral
scratch in turtles actually reconstructed rostral scratch motor rhythms
with normal interlimb (right-left HF) and intralimb (HF-HE) alternation
after unilaterally evoked scratch responses were rendered almost
completely tonic by strychnine (see Fig. 8 in Currie and Lee
1997
). Other investigators also have described
strychnine-resistant right-left alternation in fictive motor patterns
elicited by chemical (Kremer and Lev-Tov 1997
;
McPherson et al. 1994
) or electrical (Magnuson
and Trinder 1997
) stimulation of the spinal cord. The
persistence of interlimb and intralimb alternation in the presence of
strychnine implies that other transmitter systems, such as
GABAA (Cowley and Schmidt 1995
;
Kremer and Lev-Tov 1997
) and strychnine-insensitive
glycine receptors (Kuhse et al. 1990
) may contribute to
reciprocal inhibition in turtle scratch networks. Future experiments
are required to identify these transmitter systems and further assess
the role of both crossed and uncrossed reciprocal inhibition in
hindlimb rhythmogenesis during fictive scratching and other rhythmic
motor activity, including fictive locomotion (Juranek and Currie
1998
).
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ACKNOWLEDGMENTS |
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This research was supported by National Science Foundation Grant IBN-9723973 to S. N. Currie.
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FOOTNOTES |
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Address reprint requests to S. N. Currie.
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 14 September 1998; accepted in final form 10 February 1999.
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REFERENCES |
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