Department of Biology, Marquette University, Milwaukee, Wisconsin
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ABSTRACT |
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Buchanan, James T. Commissural interneurons in rhythm generation and intersegmental coupling in the lamprey spinal cord. To test the necessity of spinal commissural interneurons in the generation of the swim rhythm in lamprey, longitudinal midline cuts of the isolated spinal cord preparation were made. Fictive swimming was then induced by bath perfusion with an excitatory amino acid while recording ventral root activity. When the spinal cord preparation was cut completely along the midline into two lateral hemicords, the rhythmic activity of fictive swimming was lost, usually replaced with continuous ventral root spiking. The loss of the fictive swim rhythm was not due to nonspecific damage produced by the cut because rhythmic activity was present in split regions of spinal cord when the split region was still attached to intact cord. The quality of this persistent rhythmic activity, quantified with an autocorrelation method, declined with the distance of the split spinal segment from the remaining intact spinal cord. The deterioration of the rhythm was characterized by a lengthening of burst durations and a shortening of the interburst silent phases. This pattern of deterioration suggests a loss of rhythmic inhibitory inputs. The same pattern of rhythm deterioration was seen in preparations with the rostral end of the spinal cord cut compared with those with the caudal end cut. The results of this study indicate that commissural interneurons are necessary for the generation of the swimming rhythm in the lamprey spinal cord, and the characteristic loss of the silent interburst phases of the swimming rhythm is consistent with a loss of inhibitory commissural interneurons. The results also suggest that both descending and ascending commissural interneurons are important in the generation of the swimming rhythm. The swim rhythm that persists in the split cord while still attached to an intact portion of spinal cord is thus imposed by interneurons projecting from the intact region of cord into the split region. These projections are functionally short because rhythmic activity was lost within approximately five spinal segments from the intact region of spinal cord.
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INTRODUCTION |
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In lamprey, as in other vertebrates, the network
of neurons generating the locomotor rhythm is located within the spinal
cord. This is shown by the presence of rhythmic ventral root bursting in the isolated spinal cord when perfused with an excitatory amino acid
such as glutamate (Cohen and Wallén 1980). The
pattern of this rhythmic activity is similar to the pattern of myotomal
electromyographic activity in the swimming lamprey and is therefore
referred to as fictive swimming (Wallén and Williams
1984
). Fictive swimming is characterized by an alternation of
ventral root bursts on opposite sides of the spinal cord and a
rostral-to-caudal propagation of the bursts. As few as two to three
spinal segments taken from any rostral-caudal level can exhibit
fictive swimming, so the locomotor network is conceived of as a chain
of overlapping coupled oscillators (Cohen et al. 1992
).
A proposed model (Fig. 1A) for
the unit locomotor rhythm generator has at its core reciprocal
inhibition between commissural interneurons located on opposite sides
of the spinal cord (Buchanan 1986; Buchanan and
Grillner 1987
). This model is based on the patterns of synaptic
interactions among interneurons revealed with paired intracellular
microelectrode recordings (Buchanan 1982
;
Buchanan et al. 1989
) and is similar to a model for the swim network in the newly hatched Xenopus spinal cord
(Roberts 1990
). One characterized class of commissural
interneurons in lamprey is called CC interneurons because the main axon
of these cells projects contralaterally and caudally, although they
often also have a rostral-axonal bifurcation (Buchanan
1982
). The model also contains excitatory interneurons (EINs)
with ipsilateral axons (Buchanan et al. 1989
) and
inhibitory lateral interneurons with ipsilateral descending axons
(Rovainen 1974
).
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For the proposed network model (Fig. 1A), rhythmogenesis
originates mainly from the reciprocal inhibition between commissural interneurons on opposite sides of the cord. Activity in commissural interneurons on one side inhibits the activity of cells on the opposite
side, and when these inhibited commissural interneurons escape or are
released from inhibition (Wang and Rinzel 1992) they
become active and inhibit cells of the previously active side. A key to
the functioning of reciprocal inhibition as a rhythm generator is the
mechanism for this escape or release. Within the network model of Fig.
1A, the lateral interneurons provide a feedforward
inhibition that can terminate ipsilateral CC interneuron activity, but
other burst terminating mechanisms have been proposed such as
accumulation of calcium-activated potassium conductance (El
Manira et al. 1994
). However, for the network model of Fig. 1A, rhythmicity is lost when the outputs of commissural
interneurons are removed (Buchanan 1992
).
An alternative to the proposed network model of Fig. 1A is one in which each side of the spinal cord contains independent rhythm generators, coupled via commissural interneurons (Fig. 1B). In this model, the inhibitory commissural interneurons produce antiphasic coupling of the two sides but are not essential to rhythm generation. Rhythm generation in this model would be the result of cellular and network properties that are as yet not known in lamprey. The model of Fig. 1B predicts that removal of the outputs of commissural interneurons would not abolish rhythmic activity but would only uncouple the rhythm generators on each side of the spinal cord.
The necessity of commissural interneurons for rhythm generation has not
been clearly established in the lamprey. One approach to this question
has been to apply strychnine to the spinal cord because the output
inhibitory postsynaptic potentials of commissural interneurons are
glycinergic and are blocked by strychnine (Buchanan 1982; McPherson et al. 1994
). Application of
strychnine (5 µM) can speed fictive swimming and ultimately eliminate
rhythmicity (Grillner and Wallén 1980
;
McPherson et al. 1994
), or it can lead to synchronous
rhythmic activity on the two sides of the spinal cord (Alford
and Williams 1989
; Cohen and Harris-Warrick 1984
; Hagevik and McClellan 1994
). Lower
concentrations of strychnine (0.1-0.2 µM) can produce a slow
modulation of the swimming rhythm that maintains an alternating pattern
(McPherson et al. 1994
). Thus the results of strychnine
experiments are mixed with regard to the issue of the necessity of
commissural interneurons. One problem with strychnine application is
that it blocks almost all synaptic inhibition in the spinal cord, not
just commissural inhibition (Homma and Rovainen 1978
),
and may also block GABA receptors (Baev et al. 1992
).
Strychnine in the 1- to 10-µM range is not selective for ligand-gated
receptors but can also reduce voltage-gated sodium and potassium
currents (Shapiro et al. 1974
) and voltage-gated calcium
currents (Oyama et al. 1988
). Strychnine application may thus result in rhythmic activity that is not directly relevant to
activity in the locomotor network. The most direct test of the
necessity for commissural interneurons is to make midline cuts that
completely isolate the spinal cord into lateral halves (hemicords). It
has been reported that lateral hemicords of lamprey can show rhythmic
activity in the presence of strychnine (Grillner et al.
1986
), but again it is not clear to what extent this
strychnine-dependent rhythmic activity relies on the locomotor network.
In this study, midline cuts were made on the lamprey spinal cord to assess the necessity of commissural interneurons in the production of fictive swimming. The cuts were first done partially, beginning at one end of the spinal cord piece and extending to approximately one-half the length of the piece. This partial cut was done to determine whether the midline cut produced nonspecific damage that could block rhythmic activity. These partial midline cuts yielded preparations in which rhythmic activity was present in the split spinal cord located near the intact cord but was absent in more distant split segments. After complete isolation of the two lateral halves of the spinal cord, the rhythmic activity of fictive swimming was lost. These experiments not only demonstrate that nonspecific damage is insufficient to explain the loss of rhythmic activity but also provide insight into the lengths of the coupling signals among the locomotor rhythm generators.
The current experiments support the proposal that the commissural interneurons are necessary for the generation of the swim rhythm in lamprey and suggest that the functional lengths of the coupling connections are relatively short, on the order of approximately five segments.
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METHODS |
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The experiments were done on seven adult silver lampreys
(Ichthyomyzon unicuspis), 18-30.5 cm in length. Each animal
was anesthetized by immersion in a 0.01% solution of tricaine
(3-aminobenzoic acid ethyl ester) (Sigma) until reflexes were lost. The
animal was then decapitated, the brain was destroyed mechanically, and
a spinal cord-notochord preparation was dissected in cooled Ringer solution as previously described (Rovainen 1974). The
Ringer solution consisted of the following (in mM): 91 NaCl, 2.1 KCl,
2.6 CaCl2, 1.8 MgCl2, 4 glucose, and 20 NaHCO3. The solution was continuously bubbled with 98%
O2-2% CO2 (pH 7.4), and the preparation was
perfused at a rate of 2-4 ml/min (8-9°C).
For these experiments, the spinal cord-notochord preparations typically consisted of 22 segments (range 14-50). However, no systematic differences were observed between preparation length and the parameters measured in this paper. The spinal cord pieces came mainly from the midbody of the lamprey, with some extension into the fin region in three pieces, and some extension into the gill region in two pieces. One preparation came entirely from the fin region. Once a preparation was pinned to the Sylgard-lined floor of the experimental chamber and perfused with normal Ringer solution, a longitudinal cut was made along the midline with either the tip of a 30-gauge needle or with fine spring scissors. The cuts extended either from the caudal end (n = 5) or from the rostral end (n = 5) to ~40% of the length of the spinal cord piece (range 33-50%). For graphic convenience, the ventral root in the segment where the longitudinal cut began was numbered 0, and segments in the intact region were given sequential negative numbers, and segments in the cut region were given sequential positive numbers.
After the longitudinal midline cut was made, fictive swimming was induced by bath perfusion with N-methyl-D,L-aspartate (NMA) (0.2 to 0.3 mM) or with N-methyl-D-aspartate (NMDA) (0.15-0.2 mM). The resulting swim cycle periods ranged from 0.41 to 1.22 s, mean = 0.67 ± 0.22 (SD). Three ventral roots were monitored by placing the tips of glass suction electrodes onto ventral roots near their exit points from the spinal cord (Fig. 2, A and B). Two of the electrodes were stationary and were placed on opposite sides of the spinal cord in the intact region, and a third electrode was moved from root to root along the spinal cord. Ventral roots on one side of the cord were sampled with the roving electrode with recordings of 1-min duration each. Then, while recording from a ventral root in the split cord region located near the intact cord, a transverse cut was made at the beginning of the longitudinal midline cut on the recorded side to isolate that lateral hemicord (see Fig. 5 for a schematic drawing of these cuts). Activity of ventral roots in the isolated hemicord was then sampled. A similar procedure was then carried out on the opposite side of the cord. The ventral root recordings were band-pass filtered (50-2,000 Hz) and stored on an eight-channel DAT recorder (Biologic) for later off-line analysis. For the off-line analysis, the ventral root signals were low-pass filtered with a cutoff frequency of 1,000 Hz and digitized at 2,000 Hz with a Cambridge Electronic Design 1401 computer interface, Spike2 software (CED), and a 486 computer.
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To quantify the quality of rhythmic activity in the ventral root recordings, an autocorrelation was performed on the event times of the ventral root action potentials. For this, the action potentials were converted to event times by setting a voltage threshold so that each time the voltage rose above the threshold level an event time was recorded. This threshold value was set just above the intrinsic noise level of the ventral root recording (Fig. 2B). An autocorrelation was performed on these event times with software provided by Spike2. For each event, the numbers of events occurring in consecutive 20-ms bins were counted over a 3-s time period (150 bins); this counting procedure was repeated for all events in the 1 min of recorded activity, and the resulting counts were summed for each bin. The counts were normalized to the maximum bin count (i.e., the first bin).
The resulting autocorrelation was a decaying oscillation (Fig. 2, C-E). The period of the autocorrelation oscillation was the same as the mean cycle period of fictive swimming. The oscillations became smaller in trough-to-peak amplitude because of lower peaks and higher troughs. The fall in the peaks is due to irregularity of the cycle periods so that sequential bursts do not exactly align; the rise in the troughs is due to the presence of spikes occurring between the ventral root bursts. The degree of attenuation of the trough-to-peak amplitudes of the oscillations in the autocorrelation gives an overall measure of rhythm quality, i.e., the regularity of the swim cycle period and the degree of confinement of spiking to the bursts. Therefore, to provide a measure of the quality of rhythmic ventral root bursting, the trough-to-peak amplitude of the second peak of the autocorrelation was used (Fig. 2C). The base of the peak was defined as the mean of the minimum values of the first and second troughs, and the amplitude from this level to the peak was measured (Fig. 2, C-E). The second peak amplitude of the autocorrelation is referred to in this paper as the quality of rhythmic activity or (QRA) and could potentially range from 0 to 1.0. Actual measured values ranged from 0.01 to 0.94.
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RESULTS |
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Partial midline cuts
Spinal cord preparations were first cut along the midline from one end to ~40% of the total length of the piece. When NMDA (or NMA) was then applied to the bath, rhythmic ventral root bursting was induced. In the intact region of spinal cord, this bursting was regular with relatively few spikes occurring between bursts, as is typical of fictive swimming in nonlesioned spinal cord (Fig. 2B). In contrast, ventral root bursting was less obvious within the region of spinal cord in which the two sides of the cord were separated by the longitudinal midline cut (i.e., the split region). Although some rhythmicity was apparent, the distinction between bursts and interburst periods was less clear. To quantify the quality of rhythmic activity, autocorrelations of the action potential event times were performed, and the normalized trough-to-peak amplitudes of the second peak of the autocorrelation were measured. This amplitude is referred to as the QRA. In the particular experiment of Fig. 2, the ventral roots in the intact region of cord had QRA values of 0.76 and 0.65 for the left and right sides of the spinal cord (Fig. 2, C and D). The QRA of a ventral root within the split region of cord had a smaller amplitude of 0.22 (Fig. 2E).
Systematic recording of ventral roots in a spinal cord piece
revealed that the quality of rhythmic bursting in the split region decreased with the distance between the ventral root and the beginning segment of the longitudinal midline cut. For example, in Fig. 3 the ventral root located in the intact
cord just rostral to the beginning of a caudally directed longitudinal
midline cut (VR1) had distinct bursts and a QRA of 0.7 (Fig. 3A). In the split region, the bursts became
progressively less distinct in ventral roots located further from the
intact region of cord (compare VR+1, VR+2,
VR+3, and VR+7 in Fig. 3, A and
C). In this particular experiment, the rhythm in
VR+3 was barely apparent (QRA = 0.07), whereas at
VR+7 no rhythmic activity was detectable in the
autocorrelation. The QRA of VR+7 was 0.04, which reflects
the noise level of the autocorrelation.
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As summarized in Fig. 4, the longitudinal midline cuts reduced the quality of the swim rhythm in all preparations, and the effect was progressive with the distance of the split segment from the intact region of spinal cord. The data are divided into those preparations with the rostral end cut (Fig. 4A) and those with the caudal end cut (Fig. 4B). Although midline cuts produced reductions in rhythm quality in the intact region of both types of preparations, those with the rostral end cut generally had poorer rhythms in the intact region than did those with the caudal end cut (Fig. 4C). In spite of this difference in the quality of rhythmic activity in the intact region, the fall in rhythm quality in the split region with distance showed a similar relationship in the two preparations (Fig. 4D).
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Complete midline cuts
When the split region of spinal cord was completely separated from the intact region by making a transverse hemisection at the beginning of the longitudinal midline cut, the rhythmic activity of fictive swimming was always eliminated in the ventral roots of the isolated lateral hemicord. For example, in Fig. 5A a ventral root recording in the split region of cord (right VR+1) displayed rhythmic activity while still attached to the intact spinal cord. The rhythmicity was apparent both in the raw ventral root activity (Fig. 5A2) and in the autocorrelation (Fig. 5A3). When a transverse cut was made to completely isolate the right split region from the intact cord (Fig. 5B1), rhythmic activity was lost in the right hemicord. Again the loss of rhythmicity was apparent both in the raw ventral root activity (Fig. 5B2) and in the autocorrelation (Fig. 5B3). The same result was observed when the other one-half of the split region of cord was severed (compare Fig. 5, C and D).
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A second effect of removing the split region of cord was that the
quality of rhythmic activity in the intact region of cord tended to
improve. For example, in Fig. 5A the QRA in the intact region (left VR5) showed an improvement from 0.24 to 0.59 after the right one-half of the split region was removed (Fig. 5,
A3 vs. B3). A further improvement was observed
when the remaining left one-half was removed from 0.51 to 0.85 (Fig. 5,
C3 vs. D3).
In 6 of 19 experiments, some rhythmic activity was apparent in the isolated hemicord but was not considered to be a remnant of the fictive swim rhythm because it had a much higher frequency than the fictive swim rhythm. For example, in Fig. 6 a ventral root located on the right side in the split region (r. VR+3) showed the fictive swim rhythm while still attached to the intact cord (Fig. 6A), but after a transverse cut isolated the right hemicord (Fig. 6B) the fictive swim rhythm of 0.70-s cycle period was lost in r. VR+3. However, rhythmic activity with a cycle period of 0.22 s was at times present in both the raw ventral root activity (Fig. 6B2) and in the autocorrelation (Fig. 6B3). This faster rhythm appeared to have been present weakly before the isolation of the hemicord as an intraburst rhythm (* in Fig. 6A3).
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A summary of the effects of isolation of the lateral hemicords is shown in Fig. 7. The fictive swimming rhythm was lost in all 19 cases, and no rhythmic activity was detectable in 13 of 19 cases. In these 13, the QRA at the presumed time of the fictive swim cycle period was 0.03 ± 0.02 (± SD), which represents the noise level of the autocorrelation. In 6 of 19 cases (circles with dots in Fig. 7A), there was a higher-frequency rhythm present after the transverse cut. This rhythm was weak and had a mean QRA of 0.11 ± 0.05 (n = 6). A comparison of the cycle periods before and after the transverse cut is shown in Fig. 7B for these six preparations. The cycle period decreased from a mean of 0.74 ± 0.31 s before the cut to 0.20 ± 0.039 s in the isolated lateral hemicord.
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Figure 7C shows the increase in rhythm quality in the intact cord after removal of the split regions for all preparations. Overall, the QRA increased from 0.53 ± 0.24 to 0.77 ± 0.087 (P = 0.008, Student's t-test) after both split regions were removed. Because the preparations with the rostral end split began with poorer rhythms, they tended to show greater improvements after removal of the rostral hemicords. The preparations with the rostral ends cut improved from 0.43 to 0.80, whereas the preparations with the caudal ends cut improved from 0.63 to 0.74.
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DISCUSSION |
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Commissural interneurons and rhythm generation
These experiments demonstrated that neurons with axons crossing the midline are necessary for the generation of the swim rhythm in the lamprey spinal cord. This was shown by the loss of the swim rhythm when one side of the spinal cord was completely isolated by a longitudinal midline cut (Fig. 5). The loss of the swim rhythm in the hemicord was not due to nonspecific cell damage when the midline cut was made. This was shown by the persistence of the swim rhythm in split segments as long as the split segments were still attached to intact spinal cord. Thus, with regard to the two alternatives posed in Fig. 1, the present experiments support Fig. 1A; commissural interneurons are necessary for rhythm generation rather than simply providing antiphasic coupling of autonomous rhythm generators located on each side of the cord (Fig. 1B).
Typically, the isolated hemicords exhibited continuous ventral root
spiking in the presence of NMDA, suggesting that the midline cuts
disrupted rhythmic activity by eliminating rhythmic inhibition. Further
support for a loss of rhythmic inhibitory inputs comes from the
characteristic pattern of deterioration of the swim rhythm in spinal
cords with partial cuts. With greater distance from the intact region
of cord, the ventral root burst durations increased whereas silent
period durations decreased, suggesting that with distance there is a
progressive loss of the inhibitory signals that sculpt out the silent
inhibitory phases (Figs. 2 and 3). Inhibitory commissural interneurons,
i.e., CC interneurons, have been characterized in the lamprey spinal
cord (Buchanan 1982) and are likely to be a main source
for the rhythmic inhibition occurring during fictive swimming (Fig.
8A). Because cells with ipsilateral projections were still intact, ipsilateral inhibitory interneurons (Buchanan and Grillner 1988
) appear not to
be the main source of rhythmic inhibition during fictive swimming.
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Another important outcome of this study is that both ascending and descending commissural interneurons are important for the production of rhythmic activity. This was shown by the persistence of rhythmic activity in split regions regardless of whether the rostral or the caudal end was split (Fig. 4). This rhythmic activity would be due to ascending or descending commissural projections, respectively. Also, the quality of rhythmic activity degrades equally with distance in the two cases, suggesting that the ascending and descending commissural influences are of comparable strength and distance.
In a few cases, the isolated hemicord displayed an irregular rhythm
that had a much higher frequency than that of fictive swimming (Fig.
6). This fast rhythm was similar in frequency to intraburst rhythms
sometimes observed during fictive swimming (Fig. 6). These intraburst
rhythms may be due to intrinsic tendencies toward oscillatory membrane
potential activity in individual motoneurons, especially in the
presence of NMDA (Moore et al. 1993; Murphey et
al. 1995
). For example, in the presence of TTX, NMDA induces oscillatory membrane potentials (Wallén and Grillner
1987
). These NMDA-induced, TTX-resistant oscillations have a
cycle period of ~1 s, but often a faster oscillation (~10 Hz) is
present on the plateau phase (Tabak et al. 1995
). Even
if intrinsic cellular mechanisms are responsible, there must also be
some mechanism for synchronization, and this may be due to electrical
coupling among motoneurons (Buchanan et al. 1998
) or to
highly interconnected premotor excitatory inputs to the motoneurons
(Buchanan et al. 1989
). Similarly, rhythmic bursting in
the presence of strychnine, in which most inhibition has been blocked,
may also be due to synchronization of the depolarization of populations
of neurons followed by intrinsic repolarizing response caused by
perhaps strong activation of potassium currents.
Further support for the conclusion that commissural
interneurons are necessary for rhythm generation comes from
photoablation studies on the lamprey spinal cord (Buchanan
and McPherson 1995). In these experiments, commissural
interneurons on one side of the spinal cord were retrogradely labeled
with eosin-dextran amines. Intracellular recordings of labeled neurons
demonstrated that illumination with an argon laser depolarized the
cells and blocked the action potential within several minutes. During
fictive swimming, laser illumination of the side of the cord with the
retrogradely labeled commissural interneurons could abolish rhythmic
activity. The deterioration in the rhythm was progressive and showed a
characteristic pattern; ventral root bursts on the nonilluminated side
increased in duration whereas the interburst silent intervals
shortened; ventral root bursts on the illuminated side decreased in
duration whereas the interburst silent periods lengthened. This pattern is consistent with the loss of inhibitory commissural interneurons on
the illuminated side, resulting in less inhibitory sculpting of
motoneuron firing on the nonilluminated side and greater inhibition of
motoneurons on the illuminated side from the disinhibited commissural interneurons on the nonilluminated side.
Functional length of multisegmental signals
The fictive swim rhythm present in the split region of the cord when still attached to intact cord is imposed by rhythmic inputs coming from neurons within the intact region of cord because it is abolished when these inputs are removed (Fig. 5). As argued previously, these inputs are most likely to be from inhibitory commissural interneurons. The fall in rhythm quality in the split region with distance from the intact cord suggests that the cells in the intact region that project into the split cord have a limited length of influence (Fig. 8A). The relationship of rhythm quality versus distance may thus give an overall measure of the functional length of these multisegmental projections. Within approximately five segments, rhythmic activity was lost in the split region (Fig. 4), suggesting that after five segments the overall strengths of the projections (number of axons and their individual synaptic strengths) were too weak to modulate motoneuron firing. The functional lengths of the ascending and descending projections appear to be similar (Fig. 4D).
The distance relationship found in this work matches well the one
observed in a separate study (Buchanan et al. 1995) in
which the correlation of synaptic potentials between motoneurons
separated by various distances showed a similar fall with distance
during fictive swimming (triangles of Fig. 4D). Thus these
two methods for estimating the functional length of coupling signals
both indicate that coupling signals act over approximately five
segments. The EINs have ipsilateral axons that appear to project fewer
than five segments (Buchanan et al. 1989
). Although
these cells may contribute to the coupling observed here, the
characteristic loss of silent phases suggests that inhibitory
interneurons are of key importance and thus may be due to commissural
interneurons with relatively short projections (Ohta et al.
1991
). However, it is known that inhibitory interneurons in the
lamprey spinal cord can project many segments. For example, some
inhibitory CC interneurons have axonal projections up to 30 segments
(Buchanan 1982
), and inhibitory lateral interneurons
have axonal projections up to 50 segments (Rovainen
1974
). The results of this study do not imply that these
longer-projecting cells have no effects during fictive swimming but
suggest that the actions of projections longer than approximately five
segments are too weak to rhythmically modulate the ventral root firing.
As stated in METHODS, no relationship was found between the
overall length of the preparation and the rate of deterioration of the
rhythmic activity with distance. However, there is evidence that longer
connections do have effects during fictive swimming. For example,
modeling of cross-correlations of ventral root bursting has suggested
the presence of long connections (Mellen et al. 1995
).
In addition, partitioned bath experiments have also shown that longer
connections exist (Miller and Sigvardt 1996
;
Rovainen 1985
). In these latter experiments, activity in the middle of a spinal cord piece was blocked while allowing the two
ends of the cord to swim fictively. The nonswimming middle region could
be extended up to 20 segments with some maintained interactions between
the two ends. Although these experiments demonstrate that longer-range
connections exist, this study indicates that the local connections are
much stronger than the longer-range connections.
Influence of the split cord on the intact cord
In addition to the presence of neurons in the intact spinal
cord region that impose rhythmic activity on the split segments, it was
also clear that neurons within the split spinal cord region could
influence the activity in the intact cord. These cells would be neurons
with ipsilateral axon projections and would likely carry abnormal
signals because they originate from segments with deteriorated rhythms
(Fig. 8B). When the split regions of the spinal cord were
removed from intact cord, not only did the rhythm cease in the split
region (caused by the loss of rhythmic input from the intact region),
but the quality of rhythmic activity in the intact region improved
(caused by removal of disruptive inputs from the split region) (Figs. 5
and 7). In most cases (Figs. 4 and 7C), the rhythm in the
intact region was more disrupted when the rostral end of the spinal
cord piece was split, suggesting that descending ipsilateral neurons
have a more powerful influence on the rhythm-generating networks than
ipsilateral ascending neurons. Alternatively, the descending
projections may be longer than the ascending ones and thus carry
signals from the more distant and more disrupted segments of the split
region. This would be consistent with the results of partitioned-bath
experiments in which the measurement of the distance of projections
from an actively swimming one-half of spinal cord into the passive
one-half demonstrated that ascending neurons have shorter-range effects
during fictive swimming than do descending neurons (Dale
1986).
Comparison with other vertebrates
In comparison with other vertebrates, the lamprey spinal cord may
be an exception with regard to the necessity for commissural interneurons for rhythm generation. In the embryonic
Xenopus, midline cuts separating the halves of the brain and
spinal cord do not abolish rhythmic activity (Kahn and Roberts
1982; Soffe 1989
). After separation in the
Xenopus preparation, rhythmic activity is somewhat faster
and more variable than in the intact animal. The persistent rhythmic
activity has been postulated to be due to a combination of feedback
excitation from motoneurons to produce synchronization and recurrent
inhibition from ipsilateral collaterals of commissural interneurons
(Roberts et al. 1997
). In limbed vertebrates, a
direct comparison to lamprey is less clear. In lamprey and
Xenopus, antagonistic muscle groups are located on opposite
sides of the body, whereas in limbed vertebrates, antagonistic muscles
act on limb joints on the same side of the body. The motoneurons and interneurons associated with these antagonistic groups are also located
on the same side of the spinal cord. Thus in limbed vertebrates the
cells that would be functionally similar to commissural interneurons of
the lamprey, i.e., those providing reciprocal inhibition between antagonist neuronal groups, are not easily accessible for lesion studies as done in this study. There is evidence, however, that commissural interneurons may participate in rhythmic pattern formation in higher vertebrates. For example, in the fictive scratch rhythm of
adult turtles (Stein et al. 1995
), activity of one
hindlimb scratch network generates weak activity in the contralateral
limb scratch network, and activation of these commissural projections can in some cases be necessary for the expression of rhythmic activity
(Currie and Gonsalves 1997
). In the locomotor rhythm of
the neonatal rat spinal cord, selective pharmacological activation of
one side of the lumbar spinal cord produces rhythmic inhibition in
contralateral motoneurons, again demonstrating that commissural interneurons are involved in pattern formation (Kjaerulff and Kiehn 1997
). Thus, although commissural interneurons have a
role in rhythmic pattern formation in limbed vertebrates, the question of the necessity of reciprocal inhibition between antagonistic neuron
groups for rhythm generation remains uncertain.
Conclusions
The results of this study indicate that commissural interneurons are necessary for rhythm generation of fictive swimming in the lamprey spinal cord. It also suggests that these interneurons can impose rhythmic activity in segments beyond their origin, but this influence is limited to approximately five segments.
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ACKNOWLEDGMENTS |
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I thank D. Komorowski for assistance in some of the experiments.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-35725.
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FOOTNOTES |
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Address for reprint requests: J. T Buchanan, Dept. of Biology, Marquette University, P. O. Box 1881, Milwaukee, WI 53201-1881.
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 28 September 1998; accepted in final form 25 January 1999.
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REFERENCES |
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