Department of Neuroscience, University of California, Riverside, California 92521
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ABSTRACT |
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Juranek, Jenifer and Scott N. Currie. Electrically Evoked Fictive Swimming in the Low-Spinal Immobilized Turtle. J. Neurophysiol. 83: 146-155, 2000. Fictive swimming was elicited in low-spinal immobilized turtles by electrically stimulating the contralateral dorsolateral funiculus (cDLF) at the level of the third postcervical segment (D3). Fictive hindlimb motor output was recorded as electroneurograms (ENGs) from up to five peripheral nerves on the right side, including three knee extensors (KE; iliotibialis [IT]-KE, ambiens [AM]-KE, and femorotibialis [FT]-KE), a hip flexor (HF), and a hip extensor (HE). Quantitative analyses of burst amplitude, duty cycle and phase were used to demonstrate the close similarity of these cDLF-evoked fictive motor patterns with previous myographic recordings obtained from the corresponding hindlimb muscles during actual swimming. Fictive rostral scratching was elicited in the same animals by cutaneous stimulation of the shell bridge, anterior to the hindlimb. Fictive swim and rostral scratch motor patterns displayed similar phasing in hip and knee motor pools but differed in the relative amplitudes and durations of ENG bursts. Both motor patterns exhibited alternating HF and HE discharge, with monoarticular knee extensor (FT-KE) discharge during the late HF phase. The two motor patterns differed principally in the relative amplitudes and durations of HF and HE bursts. Swim cycles were dominated by large-amplitude, long-duration HE bursts, whereas rostral scratch cycles were dominated by large-amplitude, long-duration HF discharge. Small but significant differences were also observed during the two behaviors in the onset phase of biarticular knee extensor bursts (IT-KE and AM-KE) within each hip cycle. Finally, interactions between swim and scratch motor networks were investigated. Brief activation of the rostral scratch during an ongoing fictive swim episode could insert one or more scratch cycles into the swim motor pattern and permanently reset the burst rhythm. Similarly, brief swim stimulation could interrupt and reset an ongoing fictive rostral scratch. This shows that there are strong central interactions between swim and scratch neural networks and suggests that they may share key neural elements.
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INTRODUCTION |
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The turtle hindlimb executes a variety of
coordinated rhythmic movements, including three different forms of
locomotion (forward swimming, backpaddling, and
terrestrial walking; Lennard 1975; Lennard and Stein 1977
; Stein 1978
, 1981
)
and three site-specific forms of scratch reflex (rostral,
pocket, and caudal; Robertson et al.
1985
; Stein 1989
). Recent kinematic
analyses of hip and knee joint angles in intact turtles have found that
specific forms of two different behaviors, forward swimming and rostral
scratching, display strikingly similar intralimb coordination
(Earhart and Stein 2000
; Field and Stein
1997a
). In both behaviors, the onset-phase of knee extension
within the hip movement cycle was during hip flexion; however, although
hip flexion corresponded to the returnstroke of the
forward swim, it occurred during the powerstroke (rub of the foot against the shell) of the rostral scratch. In earlier work, a
replica of forward-swimming movements was elicited in a single hindlimb
by stimulation of the contralateral dorsolateral funiculus (cDLF) of
the midbody spinal cord in intact and low-spinal turtles
(Lennard 1985
; Lennard and Stein 1977
;
Stein and Johnstone 1986
). Similar cDLF stimulation in
the cervical cord of high-spinal immobilized turtles evoked coordinated
swimming movements in the hindlimb and forelimb on one side and
occasionally on both sides (Stein 1978
). Stein
(1981)
showed that a low-spinal turtle in which both hindlimbs
were deafferented by dorsal rhizotomy could display normal coordinated
swimming movements in response to cDLF stimulation. This result
indicates that the swim motor pattern is generated centrally by the
spinal cord.
In the present study, we extended this earlier work by demonstrating
that fictive swimming could be evoked in hindlimb muscle nerves by electrical stimulation of the cDLF in low-spinal, chemically paralyzed turtles. Stimulation of the cDLF produced coordinated rhythmic discharge in hindlimb motoneurons that resembled the electrically evoked swim electromyogram (EMG) motor patterns recorded from intact and low-spinal turtles with movement (Earhart and Stein 2000; Lennard 1975
; Lennard
1985
; Lennard and Stein 1977
; Stein and
Johnstone 1986
). Cutaneous stimulation of the lateral midbody
(shell bridge) in the same preparations produced fictive rostral
scratching. We observed interactions between fictive swim and
fictive rostral motor patterns. Brief rostral scratch stimulation could
interrupt and reset an ongoing fictive swim rhythm, and vice versa.
Interrupted motor patterns displayed smooth transitions between swim
and scratch behaviors. These findings show that swim and scratch neural
networks impinge on each other in the spinal cord and imply that they
may share at least some rhythm generating elements.
Previous experiments from our laboratory have demonstrated that
electrical stimulation of an identified cutaneous nerve can evoke
vigorous pocket scratch motor patterns in a highly reduced in vitro
preparation of the turtle spinal cord with attached hindlimb nerves
(Currie and Lee 1996). The present experiments serve as a foundation for future investigations in which both fictive scratch and fictive swim motor patterns can be elicited in the same in vitro
preparations, facilitating a cellular analysis of shared circuitry and
motor pattern selection in the turtle spinal cord. Our data were
published previously in abstract form (Juranek and Currie
1998
).
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METHODS |
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Adult red-eared turtles, (n = 12; Charles D. Sullivan, Nashville, TN), Trachemys scripta elegans,
weighing 440-775 g, were submerged in crushed ice for 2 h before
surgery to induce hypothermic anesthesia (Lennard and Stein
1977
). Turtles remained partially submerged in crushed ice
during all surgical procedures. A dorsal laminectomy was performed to
expose the second through fourth postcervical segments of the spinal
cord (D2-D4), just
posterior to the forelimb enlargement. The spinal cord was then
completely transected between postcervical segments
D2 and D3.
Surgical procedures
We prepared several hindlimb nerves for electroneurographic
(ENG) recording (see Robertson et al. 1985). Each nerve
was freed from the surrounding tissue, tied with surgical thread near
its muscle insertion, and then cut distal to the tie. ENGs were
obtained from the following nerves in all turtles: VP-HP, HR-KF, and
FT-KE. 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. FT-KE innervates the monoarticular knee extensor muscle triceps
femoris pars femorotibialis, vastus medialis. In eight turtles, ENGs
were also obtained from two biarticular knee extensor nerves, AM-KE and
IT-KE, which innervate triceps femoris pars ambiens and iliotibialis, respectively. Hereafter in the text, VP-HP is referred to as the hip
flexor (HF) and HR-KF as the hip extensor (HE) nerve.
ENG Recordings
After surgery was complete, turtles were removed from the
crushed ice, allowed to warm up to room temperature (21-24°C), and immobilized with an 8-mg/kg dose of gallamine triethiodide (Sigma, St.
Louis, MO) injected intramuscularly. Rings of warm dental wax were
molded around the holes in the dorsal carapace over the exposed
D2-D4 spinal cord and the
dissected hindlimb nerves and glued in place with Permabond adhesive.
Animals were then intubated and placed on a ventilator; respiration was
maintained throughout the experiment at a rate of 1.0-1.2 cycles/min.
The wax well surrounding the exposed spinal cord was filled with
Tris-buffered physiological saline (pH 7.6, modified from Stein
and Schild 1989). The well surrounding the dissected hindlimb
nerves was filled with mineral oil (Robertson et al.
1985
). Differential ENG recordings were obtained with bipolar
hook electrodes (100-µm silver). ENG signals were amplified and
filtered (band-pass 0.1-1.0 kHz), digitized by an eight-channel pulse
code modulation (PCM) video adapter (Vetter, Rebersburg,
PA), and stored on videotape with a voice channel and stimulus marker
for off-line analysis on a computer.
Stimulation procedures
The D3 end of the severed spinal cord was lifted up by the meninges and held in place by Gelfoam to permit a transverse view of the spinal cord gray and white matter. Concentric bipolar microelectrodes (MCE-100; Rhodes Medical Instruments, Woodland Hills, CA) were used to deliver constant-current pulses (8-24 µA, 1- or 2-ms pulses; 20-60 Hz, 5-25-s trains) to the spinal white matter, concentrating on sites in the cDLF. Preparations were always given at least a 3-min rest between successive stimulus trains. A camera lucida was attached to the stereo dissection microscope at our physiology setup. We used this during the experiment to draw the location of the electrode tip relative to the spinal cord gray and white matter for each new stimulation site.
Mechanical or electrical stimulation of cutaneous afferents was used to
evoke fictive rostral scratching. Mechanical stimulation was applied to
sites in the rostral scratch receptive field with a fire-polished glass
probe attached to the end of a hand-held force transducer (Grass FT-03,
Astro-Med, West Warrick, RI). Electrical stimulation was applied via
pin electrodes spaced 2-3 mm apart in the shell epidermis (1-ms,
10-20-V pulses; 3-50-Hz trains) (Currie and Stein
1990). Preparations rested for at least 3 min between scratch episodes.
Data analysis
ENG recordings and stimulus markers were redigitized off-line (2 kHz per channel) on a computer using a Digidata 1200 A/D converter and Axotape 2.0 software (Axon Instruments, Foster City, CA). Datapac II software was used to calculate the frequency, mean amplitude, duty cycle, and dual-referent phase of ENG bursts. Before making these calculations, digitized ENG recordings were full-wave rectified and rebinned at 100 Hz (i.e., the mean of 20 consecutive data points was calculated, so that there were 100 full-wave-rectified data points per second). Burst onsets and offsets were identified by the Datapac II program as positive- and negative-slope crossings over a user-specified threshold, respectively. All analyses were confined to swim and scratch cycles that occurred completely during the period of stimulation.
HF burst frequency was automatically calculated as the reciprocal of cycle period, measured between consecutive burst onsets. HF discharge that occurred before the first HE burst was not included in the analysis. The mean amplitude of an ENG burst was obtained by averaging each of the full-wave-rectified and rebinned (100 Hz) voltage measurements that occurred within the burst. Mean amplitudes were calculated for each of the bursts in up to five nerves (IT-KE, AM-KE, FT-KE, HF, and HE) during 5-10 episodes of fictive rostral scratching and 5-10 episodes of fictive swimming in four different turtles. Averaged values obtained for each nerve during swimming were normalized to the averaged values obtained during rostral scratching in the same turtle (Fig. 3). Duty cycle was calculated as burst duration divided by HF cycle period. For each experiment in Fig. 4, we calculated the average duty cycle for 5-10 episodes of fictive swim and 5-10 episodes of fictive rostral scratch motor patterns.
We calculated dual-referent phase values for the onsets and offsets of
knee extensor bursts (IT-KE, AM-KE, and FT-KE) within the ipsilateral
HF activity cycle. Dual-referent phase measurements are appropriate for
periodic events with variable duty cycles (Berkowitz and Stein
1994). The HF activity cycle was divided into HF-on
and HF-off periods. The onsets of HF bursts were defined by
phase values of 0.0 and 1.0. The offsets of HF bursts were defined by a
phase value of 0.5. Circular statistics were used to analyze phase
values (Batschelet 1981
; Zar 1984
). The
angle and length of the mean vector were calculated for each knee
extensor using standard trigonometric functions. The angle of the mean vector represents the average phase value on a circular scale ranging
between 0.0 and 1.0. The length of the mean vector indicates the degree
to which individual data points were concentrated along the mean
vector. We used the Rayleigh test to assess the statistical significance of the mean vector length. Thus, the Rayleigh test enabled
us to determine whether phase values were random or locked to a
particular portion of the HF activity cycle. To assess significant differences in the angles of the mean vectors between swim and scratch,
we calculated confidence intervals (99%) for each mean angle and
compared them (Batschelet 1981
; Fisher
1995
). Overlapping confidence intervals were noted as
statistically insignificant. This statistical method was preferred over
other nonparametric tests (e.g., the Watson U2
test; Batschelet 1981
), which do not discriminate
between differences in mean angle and differences in angular deviation
(circular analog of SD).
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RESULTS |
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Qualitative comparison of fictive swimming elicited by spinal cord stimulation and fictive rostral scratching evoked by shell stimulation
We elicited coordinated, cyclic motor output from hindlimb muscle nerves in low-spinal, chemically paralyzed turtles by electrically stimulating the spinal white matter with a concentric bipolar microelectrode. Trains of stimulus pulses applied to sites in the cDLF, at the anterior end of segment D3 (just posterior to the forelimb enlargement), evoked stereotyped fictive swim motor patterns in 11 of 12 preparations. One animal exhibited only tonic ENG discharge, regardless of stimulus location and pulse parameters. Nine of the 11 turtles that exhibited cDLF-evoked fictive swimming in this study also displayed vigorous fictive rostral scratching in response to electrical or mechanical shell stimulation within the rostral receptive field. This enabled us to compare fictive swim and rostral scratch motor patterns in the same animals.
Figure 1 shows examples of cDLF-evoked fictive swimming and sensory-evoked fictive rostral scratching recorded as ENGs from five different hindlimb muscle nerves in the same low-spinal immobilized turtle. Both motor patterns displayed rhythmic alternation between HF and HE activity; however, the relative amplitudes and durations of HF and HE bursts were substantially different in the two behaviors. Fictive swimming was strongly HE biased, exhibiting small amplitude, short-lasting HF bursts and large amplitude, long-lasting HE bursts (Fig. 1A). In contrast, fictive rostral scratching was highly HF biased, with large, long-lasting HF bursts and small, short-lasting HE discharge (Fig. 1B). Knee extensor bursts (IT-KE, AM-KE, and FT-KE) were also relatively small and brief during the swim, compared with the rostral scratch. The most dramatic amplitude changes occurred in the biarticular knee extensors, IT-KE and AM-KE. In four of eight turtles, IT-KE and AM-KE bursts were so reduced during fictive swim episodes that they either disappeared entirely or were too small to measure. The timing of KE discharge within the hip flexor-extensor cycles was similar during swim and rostral scratch motor patterns; in both cases, knee extensor bursts occurred largely during the latter part of each HF burst.
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We did not systematically map the motor output elicited by electrical stimulation in all areas of the contralateral and ipsilateral white matter. However, at the beginning of most experiments, we sampled several sites within both the cDLF and ipsilateral DLF (iDLF) before concentrating on a few effective sites in the cDLF. Not all cDLF sites were equally effective for eliciting a swim motor pattern; however, we never observed motor patterns that resembled fictive swim activity while stimulating sites outside the cDLF. iDLF stimulation produced a wide variety of rhythmic and nonrhythmic motor responses, but none of these displayed the HE-biased hip alternation and knee extensor timing characteristic of fictive swimming. Rhythmic responses elicited by iDLF stimulation typically exhibited large-amplitude HF bursts and small-amplitude HE bursts, compared with cDLF-evoked motor patterns (data not shown). During iDLF-evoked motor activity, HF and HE bursts were often partially coactive, in contrast to the distinct HF-HE alternation evoked by cDLF stimuli. Both contralateral and ipsilateral DLF stimulation could sometimes trigger fictive scratch cycles (identified based on FT-KE timing within the hip cycle and relative amplitudes of HF and HE bursts), but these virtually always occurred as "off-responses" after cessation of the DLF stimulus train, rather than during stimulation (see description of Fig. 2, below).
The burst frequency of fictive swim motor patterns could be controlled by the frequency or amplitude (current) of stimulus pulses applied to the cDLF. In one experiment, stimulus frequency was increased in 5-Hz steps from 20 to 60 Hz; within this range, HF burst frequencies exhibited a twofold increase, from 0.21 to 0.45 Hz. When stimulus amplitude was increased from 8 to 16 µA in this preparation, the frequency of HF bursting changed from 0.27 to 0.41 Hz. In other experiments, we also found that increasing the duration of stimulus pulses (e.g., from 0.1 to 0.2 ms) could increase fictive swim frequency when other stimulus parameters were held constant (data not shown).
Electrically evoked fictive swimming was "gated" by the cDLF stimulus train. Swim motor output always ceased within 1 cycle after stimulus offset (Fig. 2, A and B). In contrast, fictive rostral scratching typically continued for one or more full cycles after an electrical or mechanical shell stimulus; scratch afterdischarge was especially pronounced after brief (nonfatiguing) shell stimulation (Fig 2, C and D). The occurrence of prolonged afterdischarge during scratch reflex was an additional feature that differentiated rostral scratch from cDLF-evoked swim motor patterns. In some preparations (n = 8), we observed cyclic motor activity that continued for up to several seconds after cessation of a cDLF stimulus; however, this afterdischarge never resembled fictive swimming. When cDLF-evoked afterdischarge occurred, it usually exhibited the timing and burst amplitude characteristics of the rostral scratch reflex. In a few cases, the motor activity could not be identified.
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Quantitative analyses of fictive swim and rostral scratch motor patterns
We performed quantitative analyses on hindlimb motor patterns from the four turtles that displayed both robust cDLF-evoked fictive swimming and excellent fictive rostral scratching elicited by electrical stimulation of the shell bridge. In three of these four turtles, ENGs were recorded from five hindlimb nerves (IT-KE, AM-KE, FT-KE, HF, and HE) on the right side; in the remaining turtle, ENGS were recorded from four nerves (AM-KE, FT-KE, HF, and HE). We used rectified and rebinned ENG data (see METHODS) for all measurements of mean amplitude, duty cycle, and phase. For each turtle, we adjusted the electrical stimulus parameters of the cDLF train to elicit swim frequencies that were comparable to rostral scratch frequencies generated in the same turtle (±0.1 Hz).
MEAN BURST AMPLITUDES. Figure 3 compares the mean burst amplitudes of hindlimb nerve recordings for fictive swim and fictive rostral scratch motor patterns. For each nerve, the mean amplitude obtained from swim episodes was normalized to the mean amplitude from rostral scratch episodes in the same animal. The results clearly illustrate the relatively large-amplitude HE bursts and small-amplitude HF bursts characteristic of fictive swimming, compared with fictive rostral scratch motor patterns. The mean amplitudes of HE discharge during fictive swimming were 128-150% of the values obtained during the fictive rostral scratch. In contrast, HF, IT-KE, AM-KE, and FT-KE bursts nearly always had significantly lower mean amplitudes during fictive swimming compared with rostral scratch episodes. The mean amplitudes of HF bursts during fictive swimming were 47-57% of their respective rostral scratch values. For IT-KE, AM-KE, and FT-KE bursts, mean amplitudes during swimming ranged between 47-70% of rostral scratch measurements. During one experiment (3-3-98), IT-KE activity was recorded but was so reduced during fictive swimming that it could not be measured. This same preparation was the only case in which FT-KE bursts did not exhibit significantly lower amplitudes during swimming, relative to the rostral scratch.
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DUTY CYCLES. Swim and rostral scratch motor patterns exhibited large and consistent differences in duty cycle measurements (duty cycle = burst duration as a fraction of cycle period) for hip and knee ENG bursts (Fig. 4). Fictive swimming was highly HE-biased, displaying a consistent pattern of short HF and long HE bursts. The range of average HF duty cycles during fictive swimming was 0.17-0.33, whereas the range of HE values was 0.66-0.83. Across all four experiments, the ratio of average HF:HE duty cycles ranged between 0.2 and 0.5 for swim motor patterns. In contrast, fictive rostral scratch episodes were strongly HF-biased, exhibiting long HF and short HE bursts. The range of average HF duty cycles during rostral scratching was 0.78-0.86, whereas the range of HE values was 0.14-0.22. The ratio of average HF:HE duty cycles varied from 3.5 to 6.2 for rostral scratch motor patterns. Knee extensor duty cycles, similar to HF cycles, were significantly shorter during swim motor patterns, compared with the rostral scratch. In two experiments, the average IT-KE duty cycles were 0.12 and 0.19 during swimming, but 0.82 and 0.81 during rostral scratching in the same preparations (Fig. 4, A and B). Across all four experiments, the range of AM-KE and FT-KE duty cycles was 0.11-0.2 during swimming and 0.34-0.56 during rostral scratching (Fig. 4, A-D).
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PHASE OF KNEE EXTENSOR ACTIVITY.
We calculated the average phase values (see METHODS) for
the onsets and offsets of knee extensor bursts (IT-KE, AM-KE, and FT-KE) relative to two referents in the HF cycle, comparing these values for fictive swim and rostral scratch motor patterns (Fig. 5). In the present experiments, we found
that fictive swimming and rostral scratching both exhibited
FT-KE discharge that began during the middle to late part of the
HF-on period (phase 0.25) and ended near
the beginning of the HF-off period (phase
0.5). Therefore, fictive swim and rostral scratch motor patterns
could not be distinguished based on the timing of FT-KE bursts. Figure 5 shows that in three of four turtles, the onset phase of
FT-KE discharge was not significantly different in our fictive swim and
rostral scratch motor patterns (Fig. 5, A-C). The
offset phase of FT-KE bursts, as well as IT-KE and AM-KE
discharge, was also not significantly different in three of four
experiments (Fig. 5, B-D). The only consistent phase
differences we found between fictive swim and rostral scratch episodes
was in the onset timing of the biarticular knee extensors, IT-KE and
AM-KE. Both IT-KE and AM-KE exhibited significantly later onsets with
respect to the HF cycle during fictive swimming, compared with rostral
scratching (Fig. 5, A-C; P < 0.01; 2 of 2 turtles with IT-KE, 3 of 4 turtles with AM-KE). This
difference was especially striking in IT-KE, which began to fire
after HF-onset during swim cycles (i.e., >0.0 phase), but
before HF-onset during rostral scratch cycles (i.e., <0.0
phase; Fig. 5, A and B; see also Fig.
1).
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Interactions between fictive swim and rostral scratch motor patterns
In Fig. 6, we show that brief stimulation of rostral scratch during an ongoing fictive swim motor pattern could interrupt and permanently reset the swim rhythm. We observed such interactions in five turtles. In this particular animal, control fictive swim episodes elicited by cDLF stimulation displayed HF-HE alternation in which the HF bursts were unusually weak and there was virtually no KE activity (Fig. 6A). A brief electrical (Fig. 6B) or mechanical (Fig. 6D) shell stimulus applied to the ipsilateral rostral scratch receptive field elicited a partial rostral scratch cycle in this preparation while it was at rest (rest = no ongoing stimulation or motor activity). These control scratch responses exhibited prolonged HF discharge that peaked shortly after stimulus offset, with accompanying KE bursts, and then decayed slowly over many seconds. While this turtle was at rest, such brief shell stimuli were insufficient to evoke full rostral scratch cycles with alternating HF and HE bursts. Figure 6, C and E shows that similar brief stimulation of rostral scratch during fictive swim activity could insert one or more rostral scratch cycles into the swim motor pattern and reset the timing of motor bursts during the remainder of the cDLF stimulus train. In both cases (Fig. 6, C and E), the scratch stimulus was delivered during the HE phase of the swim cycle, abruptly terminating the HE burst and producing a phase-advance reset of the motor rhythm. In Fig. 6C, the electrical shell stimulus inserted a single cycle of rostral scratch, characterized by large-amplitude HF and KE bursts, into the swim motor pattern. In Fig. 6E, the mechanical shell stimulus evoked three full rostral scratch cycles. That the rostral scratch responses elicited during swim activity (Fig. 6, C and E) displayed normal HF-HE alternation, whereas the control rostral scratch responses (Fig. 6, B and D) did not suggests that the HF-biased excitation produced by rostral scratch stimulation combined with the HE-biased excitation elicited by cDLF swim stimulation to generate the alternating discharge. This result provides support for the hypothesis that swim and rostral scratch networks contain at least some shared interneuronal elements.
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In one turtle, we performed the reverse experiment of briefly stimulating the cDLF to activate fictive swimming during ongoing rostral scratch motor patterns (Fig. 7). Figure 7A shows a control rostral scratch episode in this turtle, elicited by electrical stimulation of the shell in the rostral receptive field. In Fig. 7B, we delivered a short stimulus train to the cDLF during an ongoing rostral scratch. The onset of the cDLF stimulus occurred during the HF phase of the scratch, terminating the HF burst and eliciting more than two full cycles of fictive swimming. After the offset of cDLF stimulation, the preparation ceased producing swim cycles and reverted to the rostral scratch motor pattern. Note that all three knee extensors were strongly active during the control scratch episode (Fig. 7A), but their amplitudes and durations were greatly reduced during the period of cDLF-evoked swim activity (Fig. 7B).
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DISCUSSION |
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We demonstrated that fictive swimming can be elicited
in turtle hindlimb muscle nerves by electrical stimulation of the cDLF in low-spinal, chemically paralyzed preparations. Stimulation of the
cDLF produced coordinated rhythmic discharge in hindlimb motoneurons
that resembled the electrically evoked swim EMG motor patterns recorded
from intact and low-spinal turtles with movement (Lennard
1975; Lennard 1985
; Lennard and Stein
1977
; Stein and Johnstone 1986
). Cutaneous
stimulation of the lateral midbody (shell bridge) in the same animals
produced fictive rostral scratching. Quantitative analyses
of fictive swim and rostral scratch episodes demonstrated largely
similar phasing in hip and knee motor pools, but distinct differences
in the amplitudes and relative durations (duty cycles) of ENG
discharge; identical observations were made earlier by Stein and
Johnstone (1986)
for EMG recordings in moving preparations. We
also observed interactions between swim and rostral scratch networks.
Brief rostral scratch stimulation could interrupt and permanently reset
an ongoing fictive swim rhythm, and vice versa. Interrupted motor
patterns displayed smooth transitions between swim and scratch cycles.
These findings show that swim and scratch neural networks impinged on
each other in the spinal cord and imply that they may share at least
some rhythm-generating elements.
Comparison of fictive and actual swim motor patterns
Previous investigations in intact and low-spinal
turtles demonstrated that electrical stimulation of the cDLF could
produce rhythmic hindlimb movements that resembled forward swimming
(Lennard 1975; Lennard 1985
;
Lennard and Stein 1977
; Stein 1981
;
Stein and Johnstone 1986
). Similar stimulation of cDLF
sites in the cervical spinal cord of high-spinal immobilized turtle
preparations elicited out-of-phase, coordinated swimming movements in
the forelimb and hindlimb on one side (Stein 1978
).
Electrical stimulation in the spinal DLF has also been shown to elicit
fictive swimming activity in the contralateral ventral roots of the
stingray (Williams et al. 1984
) and is well known to
evoke stepping movements in high-spinal (Kazennikov et al.
1983
; Sherrington 1910
) and low-spinal (Grillner and Zangger 1979
) cats. It was suggested by
Lennard and Stein (1977)
that cDLF stimulation in
turtles might activate descending reticulospinal axons to produce
locomotor movements. Reticulospinal axons are likely to contribute most
descending fibers in the lateral and ventral funiculi of reptilian
spinal cords (Kusuma et al. 1979
; ten Donkelaar
1976a
,b
).
Experiments with moving preparations described cDLF-evoked swim motor
patterns based on EMG recordings from 1) FT-KE and HE muscles alone (Lennard 1975; Lennard and Stein
1977
; Stein 1978
); 2) FT-KE, HF,
and HE muscles (Stein and Johnstone 1986
); or
3) AM-KE, FT-KE, HF, and HE muscles (Lennard
1985
). Lennard (1975)
found that monitoring
activity in just the FT-KE and HE muscles was sufficient to
discriminate forward swimming from other locomotor behaviors, such as
backpaddling and terrestrial walking. EMGs recorded during
forward swimming consistently exhibited brief FT-KE
activity immediately followed by intense, long-lasting HE discharge
(Lennard 1975
; Lennard and Stein 1977
;
Stein 1978
). In contrast, EMGs obtained during
spontaneous backpaddling in intact turtles displayed
intense, long-lasting FT-KE activity followed by weak, short-lasting HE
discharge (Lennard 1975
; Lennard and Stein
1977
). Differences between forward swim and terrestrial walking motor patterns were even more pronounced
(Lennard 1975
). Walking motor patterns reliably
exhibited double bursting in the FT-KE muscle, the two bursts occurring
at the onset and offset of HE discharge. Later experiments, in which
KE, HF, and HE muscles were all recorded, showed that cDLF-elicited
swimming was reliably characterized by 1) alternating HF
and HE bursts (Lennard 1985
; Stein and Johnstone
1986
), 2) asymmetric hip cycles dominated by
large-amplitude, long-duration HE bursts, with comparatively weak,
short-duration HF bursts (Stein and Johnstone 1986
), and 3) weak AM-KE and FT-KE discharge during the latter part
of each HF burst (Lennard 1985
). In the present
experiments, we found that fictive cDLF-evoked motor patterns displayed
each of these identifying characteristics. These fictive
swim motor patterns could only be evoked by electrical
stimulation of the cDLF and never occurred spontaneously or in response
to sensory (shell) stimulation.
The electrical stimulation that we used to elicit fictive swimming in
this study was similar in several respects to the stimulation used by
Lennard and Stein (1977) to evoke actual swimming in
turtles with movement. First, in both studies, effective stimulation
sites for eliciting the swim motor pattern were located in the
contralateral DLF at the anterior end of segment D3.
Stimulation applied outside this area (e.g., the ipsilateral DLF) did
not elicit swim motor patterns. Second, moving and fictive swim motor
patterns were both "gated" by the stimulus train. In other words,
rhythmic swim activity continued only so long as the cDLF stimulus was
maintained (e.g., Figs. 2, A and B and
6A in the present study; Fig. 9 in Lennard and
Stein 1977
). Third, increasing either the frequency or
amplitude of cDLF stimulus pulses caused a proportional increase in
swim cycle frequency in moving and fictive preparations. The range of
effective stimulus frequencies (20-60 Hz) and current amplitudes
(8-16 µA) that we observed in fictive preparations overlapped the
stimulus parameters noted by Lennard and Stein (1977)
in
moving animals (15-50 Hz and 11-19 µA; their Fig. 5). Fourth,
cDLF-evoked swimming could override ongoing motor activity, such as
spontaneous backpaddling movements (Figure 10 of Lennard and
Stein 1977
) or fictive rostral scratching (Fig. 6 of the
present study).
We observed relatively low burst frequencies during cDLF-evoked fictive
swimming (0.2-0.5 Hz), compared with the frequencies that were
previously reported for actual cDLF-evoked swimming in moving
preparations (0.5-1.6 Hz; Figs. 5 and 6 in Lennard and Stein
1977). Similar reductions in cycle frequency have been observed in deafferented vertebrate preparations (chick hatching and
walking: Bekoff et al. 1987
), isolated
vertebrate CNS preparations (mudpuppy walking:
Wheatley et al. 1992
; lamprey swimming:
Cohen 1995
and personal communication), and deafferented
invertebrate preparations (locust flight: Wilson
1961
; Pearson and Wolf 1987
; locust
grooming: Berkowitz and Laurent 1996
) compared
with the intact animals. It has been suggested that the higher cycle
frequencies observed in some intact vertebrate preparations with
movement may result from an excitatory influence of movement-related
afferent feedback (Bekoff et al. 1987
).
Comparison of fictive swim and fictive rostral scratch motor patterns
Our ENG recordings show that fictive swim and rostral scratch
motor patterns displayed similar synergies between hip and knee motor
pools but strikingly different cycle asymmetries. Stein and
Johnstone (1986) described an HF-dominated EMG motor pattern during rostral-scratching movements and an HE-dominated motor pattern
during forward-swimming movements. Our nerve recordings complement
these earlier EMG experiments and extend the preliminary descriptions
of those authors to a quantitative level. The mean amplitudes of HE
bursts were significantly greater during fictive swimming compared than
during rostral scratching (Fig. 3). In contrast, HF, IT-KE, AM-KE, and
FT-KE bursts all had significantly smaller amplitudes during fictive
swimming than during rostral scratch. Comparable differences were also
noted in burst duty cycles during the two behaviors (Fig. 4). During
fictive swimming, the mean ratios of HF-HE duty cycles ranged from 0.2 to 0.5; for rostral scratching, these ratios ranged between 3.5 and
6.2. As first proposed by Stein and Johnstone (1986)
,
these differences are not unexpected, given that the powerstroke phase
of rostral scratching is during hip flexion, whereas the powerstroke of
forward swimming is during hip extension.
We used dual-referent phase analysis to assess the onset and offset
timing of knee extensor bursts relative to the hip cycle during fictive
swim and rostral scratch motor patterns. Our results confirmed and
extended the qualitative observations made by Stein and
Johnstone (1986) for EMG recordings in moving animals. We found
that bursts of activity from the monoarticular knee extensor motor
pool, FT-KE, did not significantly change their phasing between rostral
scratch and forward swim motor patterns. In both cases, FT-KE discharge
occurred during the latter part of each HF burst. In contrast to this
similarity, FT-KE has been shown to display distinctly different timing
in each of the three forms of fictive scratch reflex (rostral, pocket,
and caudal) (Robertson et al. 1985
; Stein
1989
). FT-KE bursts occur during HF in the rostral scratch,
during HE in the pocket scratch, and just after HE but before HF during
the caudal scratch. Therefore unlike different forms of turtle
scratching, rostral scratch and forward swim motor patterns cannot be
discriminated based on FT-KE timing within the hip activity cycle.
However, the onset phase of the biarticular knee extensors, IT-KE and
AM-KE, may help to discriminate between the forward swim and rostral
scratch in some preparations. IT-KE, in particular, displayed distinct
timing differences in the two behaviors. In two different experiments,
IT-KE became active before HF during the rostral scratch,
but after HF during the swim (Fig. 5, A and
B). It remains to be seen whether these differences in IT-KE
timing are also observed during EMG recordings from moving preparations.
Shared circuitry between swim and scratch networks
Our reset experiments demonstrate strong central interactions
between forward swim and rostral scratch neural networks. We found that
shell stimulation could insert rostral scratch cycles into an ongoing
fictive swim motor pattern and permanently reset the swim rhythm (Fig.
6); conversely, cDLF stimulation could insert swim cycles into an
ongoing fictive rostral scratch episode and reset the scratch rhythm
(Fig. 7). These data expand on earlier observations made by
Stein (1981) in an extended abstract. Stein described a
low-spinal turtle with deafferented hindlimbs in which DLF-evoked
swimming movements were reset by a brief rostral scratch reflex. Those
observations, combined with our present data from immobilized animals,
show that such interruptions result from central interactions between
swim and rostral scratch neural networks, rather than from
movement-related sensory feedback. It is possible that these
interactions are mediated to some degree by shared interneurons that
participate in both motor patterns; however, the existence of such
cells has yet to be demonstrated. The concept that common neural
circuitry contributes to both the forward swim and rostral scratch is
supported by the similar knee-hip timing in both motor patterns
(Earhart and Stein 2000
; Stein and Johnstone 1986
; Fig. 5 of the present study). Furthermore, recent
observations have shown that simultaneous stimulation of the cDLF and
rostral cutaneous afferents on the shell bridge can elicit coordinated hybrid motor patterns with characteristics of both the forward swim and
rostral scratch (Earhart and Stein 2000
).
As demonstrated in several invertebrate systems, separate sensory or
neuromodulatory inputs can reconfigure a single pattern-generating network to carry out multiple behavioral functions
(Harris-Warrick and Marder 1991). However, the existence
or extent of shared neuronal elements underlying different rhythmic
movements has yet to be conclusively demonstrated in a vertebrate
system (Marder and Calabrese 1996
). Shared neuronal
elements have been proposed to mediate walking and scratching in dogs
(Sherrington 1906a
,b
) and cats (Berkinblit et al.
1978
; Gelfand et al. 1988
), walking and paw shake in cats (Carter and Smith 1986
; Smith et
al. 1986
), and the distinct hindlimb motor rhythms induced by
different neurochemicals in neonatal rat spinal cords (Cowley
and Schmidt 1994
; Kiehn and Kjærulff 1996
).
Among nonmammalian vertebrates, overlapping neural networks may
underlie walking and hatching in chicks (Bekoff et al.
1987
), swimming and struggling in frog larvae (Soffe
1993
), swimming and fast-escape responses in teleost fish
(Svoboda and Fetcho 1996
), and different forms of
scratching in the turtle (Berkowitz and Stein 1994
). A
primary goal of our future studies will be to assess the extent of
shared interneuronal circuitry between swim and scratch central pattern
generators in the turtle hindlimb enlargement. We have developed an in
vitro preparation of the turtle spinal cord with attached hindlimb
nerves that expresses fictive pocket scratch motor patterns in response
to electrical stimulation of an identified cutaneous nerve
(Currie 1999
; Currie and Lee 1996
).
Experiments are currently under way to determine whether a modified
version of this reduced preparation can generate both sensory-evoked
fictive scratching and cDLF-evoked fictive swimming. If so, the in
vitro approach could permit prolonged intracellular recording from
hindlimb interneurons during electrically evoked fictive scratch and
swim motor patterns to directly address the issue of shared CPG circuitry.
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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 6 November 1998; accepted in final form 16 September 1999.
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REFERENCES |
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