Neurobiology, Physiology, and Behavior, University of California, Davis, California 95616-8519
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ABSTRACT |
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Namba, Hisaaki and Brian Mulloney. Coordination of limb movements: three types of intersegmental interneurons in the swimmeret system and their responses to changes in excitation. During forward locomotion, the movements of swimmerets on different segments of the crayfish abdomen are coordinated so that more posterior swimmerets lead their anterior neighbors by ~25%. This coordination is accomplished by mechanisms within the abdominal nerve cord. Here we describe three different types of intersegmental swimmeret interneurons that are necessary and sufficient to accomplish this coordination. These interneurons could be identified both by their structures within their home ganglion and by their physiological properties. These interneurons occur as bilateral pairs in each ganglion that innervates swimmerets, and their axons traverse the minuscule tract (MnT) of their home ganglion before leaving to project to neighboring ganglia. Two types, ASCE and ASCL, projected an axon anteriorly; the third type, DSC, projected posteriorly. Each type fires a burst of impulses starting at a different phase of the swimmeret cycle in its home ganglion. In active preparations, excitation of individual ASCE or DSC interneurons at different phases in the cycle affected the timing of the next cycle in the interneuron's target ganglion. The axons of these interneurons that projected between two ganglia ran close together, and their firing often could be recorded by the same electrode. Experiments in which either this tract or the rest of the intersegmental connectives was cut bilaterally showed that these interneurons were both necessary and sufficient for coordination of neighboring swimmerets. When the level of excitation of the swimmeret system was increased by bath application of carbachol, the period of the system's cycle shortened, but the characteristic phase difference within and between ganglia was preserved. Each of these interneurons responded to this increase in excitation by increasing the frequency of impulses within each burst, but the phases and relative durations of their bursts did not change, and their activity remained coordinated with the cycle in their home ganglion. The decrease in duration of each burst was matched to the increase in impulse frequency within the burst so that the mean numbers of impulses per burst did not change significantly despite a threefold change in period. These three types of interneurons appear to form a concatenated intersegmental coordinating circuit that imposes a particular intersegmental phase on the local pattern generating modules innervating each swimmeret. This circuit is asymmetric, and forces posterior segments to lead each cycle of output.
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INTRODUCTION |
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Animals that use limbs to walk or swim do so by
moving those limbs periodically in a coordinated pattern. In both
arthropods and vertebrates, these rhythmic movements of each limb are
driven by local central pattern-generating circuits. These local
circuits are coordinated by interneurons the activities of which
produce the particular phase differences between neighboring limbs that are characteristic of the animal's gaits. Within a local circuit, pattern-generating interneurons can be nonspiking, and their synaptic transmission can be graded (Paul and Mulloney 1985a,b
;
Pearson and Fourtner 1975
). Communication between
circuits, however, depends on impulses in axons of coordinating
interneurons and on spike-mediated synaptic transmission to the targets
of these interneurons. The temporal patterns of impulses in these axons
contain all the information that coordinates movements of different
limbs. What are these firing patterns, what mechanisms generate these
patterns, and how do they change if the animal alters the period or
force of its movements? We have approached these questions by
investigating neurons that coordinate swimmeret movements.
The swimmerets of crayfish are a set of four paired limbs on the
abdomen that beat periodically to propel the animal forward when it is
swimming. The abdominal nervous system can produce the normal motor
program that drives these movements even when isolated from more
anterior parts of the CNS and from all sensory feedback (Hughes
and Wiersma 1960; Ikeda and Wiersma 1964
), so the swimmeret system provides an opportunity to investigate central neural mechanisms of intersegmental coordination. Each pair of swimmerets is innervated by an abdominal ganglion located in the same
segment. Within this ganglion, each swimmeret has its own local
pattern-generating module that produces alternating bursts of impulses
in its own set of power-stroke (PS) and return-stroke (RS) motor
neurons (Murchison et al. 1993
). There is no segmental gradient of excitability in these modules (Mulloney
1997
). The movements of swimmerets on each segment differ in
phase from their neighbors by ~25%, independent of beat frequency
(Braun and Mulloney 1993
; Mulloney 1997
).
Any two neighboring abdominal ganglia will generate coordinated
swimmeret motor output the phases of which are not significantly
different from those produced by the intact nervous system (Paul
and Mulloney 1986
). Thus the information conducted between
neighboring ganglia seems to be necessary and sufficient for
coordinated swimmeret movements. What neurons conduct this information?
Swimmeret modules in different segments appear to be connected by
"coordinating fibers" that project through the connectives linking neighboring ganglia (Stein 1971; Wiersma
and Hughes 1961
) to form an intersegmental coordinating circuit
(Paul and Mulloney 1986
). These units fire bursts of
impulses during either the PS phase or the RS phase of activity in
their home ganglion (Stein 1971
; Wiersma and
Hughes 1961
). These axons seem to originate in bilateral pairs
in each abdominal ganglion and to project either anteriorly, in the
ascending direction, or posteriorly, in the descending direction, at
least to the next neighboring ganglion (Skinner and Mulloney
1998
; Stein 1971
). A volley of impulses in these
ascending units affects the timing of swimmeret motor activity in the
axon's target ganglion (Stein 1971
, 1974
). Although each interganglionic connective contains thousands of axons, the numbers of these coordinating fibers that have been described are
small, and their locations in the connectives are the same from animal
to animal. The axons of certain of these intersegmental interneurons
also run in the minuscule tract (MnT) (Skinner 1985a
) of
the ganglion in which they originate (Mulloney et al.
1993
; Paul and Mulloney 1986
). To begin a
cellular analysis of these intersegmental coordinating mechanisms, we
have revisited these MnT interneurons.
Here we describe and name three types of coordinating interneurons that
originated in each abdominal ganglion that innervates functional
swimmerets. The structures and physiological properties of these
interneurons were distinct from those of command neurons or local
pattern-generating interneurons (Acevedo et al. 1994; Paul and Mulloney 1986
). The firing of these
interneurons could be recorded in MnT, and every MnT unit that fired in
phase with swimmeret activity proved to be an intersegmental
interneuron. These MnT interneurons sent axons unilaterally through the
medial ascending and medial descending tracts of the intersegmental
connectives to targets in neighboring ganglia (Stein
1971
). Experimental perturbation of individual MnT interneurons
by steps of depolarizing currents changed the timing of the next cycle
of output from their targets modules. Severing either these
intersegmental tracts or the rest of the connectives except these
tracts showed that these axons were necessary and sufficient to
maintain normal intersegmental coordination.
We also describe the responses of these interneurons to changes in excitation of the system. When excitation increased and the period of the motor pattern shortened, the phases and the relative durations (duration/period) of bursts of impulses in each of these interneurons did not change. As period shortened, they fired impulses at higher frequencies during each burst, but the durations of these bursts decreased proportionately, so the mean numbers of impulses per burst also did not change. Thus these three types of interneurons export to neighboring ganglia detailed information about the activity of the module in which they originate, cycle by cycle. This information is the basis of normal coordination of limb movements on different segments of the abdomen.
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METHODS |
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Crayfish, Pacifastacus leniusculus, were obtained from local suppliers and kept in freshwater aquaria at 15°C. Animals were anesthetized by chilling on ice. They then were exsanguinated by perfusing physiological saline into a wound created by removing one of the claws. The posterior part of the ventral nerve cord, including thoracic ganglia 4 and 5 and all six abdominal ganglia (A1, A2, ..., A6), was removed and pinned dorsal side up in a dish lined with silicone elastomer (Sylgard; Dow Corning, San Francisco, CA) and bathed in saline. The normal saline solutions contained (in mM) 195 NaCl, 5.36 KCl, 2.6 MgCl2, 13.5 CaCl2, and 10 Tris-maleate buffer, at pH 7.4. In most experiments used to observe MnT activity, preparations spontaneously expressed coordinated swimmeret motor patterns at the outset.
Neurobiotin staining
Axons of coordinating interneurons were backfilled with
Neurobiotin (Vector) through the cut ends of axon bundles stripped from
the medial-dorsal region of the interganglionic connectives. Once
filling was complete, ganglia were fixed >2 h in 4% paraformaldehyde in PBS (Acevedo et al. 1994; Sherff and Mulloney
1996
). Fixed ganglia were washed in Dulbecco's PBS (Sigma),
dehydrated to 95% ethanol to increase permeability, and then
rehydrated to PBS. To visualize the Neurobiotin, ganglia were
preincubated with 0.3% reduced Triton-X 100 in PBS, and incubated for
14-18 h with Cy3-streptavidin (1:200; Jackson Immunoresearch) or
horseradish peroxidase (HRP)-streptavidin (1:300; Amersham, Piscataway,
NJ) in 0.3% reduced Triton X-100 in PBS. HRP preparations were
developed with diaminobenzidine (DAB), using the protocol
detailed in Sherff and Mulloney (1997)
. Finally, labeled
ganglia were dehydrated, cleared, and studied as whole mounts.
Electrophysiology
Each swimmeret is innervated by one nerve, N1, which splits into
an anterior and posterior branch (Sherff and Mulloney
1997). The axons of RS motor neurons exit the ganglion through
the anterior branch; axons of PS motor neurons exit through the
posterior branch. Action potentials of PS and RS motor neurons were
recorded separately with extracellular pin electrodes on these branches
(Fig. 1A).
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Skinner (1985a) first described and named the MnT in
these abdominal ganglia and introduced the abbreviation "MT" for
it; we have substituted the abbreviation MnT here to avoid inevitable confusion with a visual area in the cortex of a minor family of chordates. To record from axons in MnT, we removed the sheath from the
dorsal side of a ganglion and placed conventional suction electrodes
(80-100 µm ID) on the dorsal surface of the lateral giant axon (LG)
near the anterior border of the lateral neuropil, LN (Fig. 1). To
confirm that these MnT units were intersegmental interneurons, another
suction electrode (Fig. 1A) was placed on the medial surface
of the desheathed hemiconnective at area 76 and area 78 (Wiersma
and Hughes 1961
).
Glass microelectrodes were filled with 2.5% Neurobiotin in 1 M KCl.
Their resistances were 30-50 M. Signals from intracellular recordings were amplified with an Axoclamp 2B preamplifier (Axon Instruments, Foster City, CA). Both extracellular and intracellular recordings were collected on VCR tape, using a Neuro-Corder 886 (Neurodata Instruments). Recordings were transferred to a computer for
analysis with Axoscope (Axon Instruments).
Surgical interruption of regions of the interganglionic connectives
To block the flow of information between two neighboring ganglia, we desheathed and separated the two hemiconnectives as we would for suction-electrode recording but then separated the dorsal-medial region (the medial parts of area 76 and area 78) that contained axons of coordinating interneurons. Once these regions were separated from the rest of the connectives, either they or the rest of the connectives were cut with fine scissors.
At the end of each of these cutting experiments, the preparation was
fixed, stained with osmium ethyl-gallate, embedded in Spurr's resin,
and sectioned for light microscopy (Leise et al. 1986).
The extent of each lesion was assessed by comparing transverse sections
of the spared region of the connective with sections of undamaged
regions of the same connective.
Perfusion procedures
To change the level of excitation in the swimmeret system
(Mulloney 1997), we superfused the isolated abdominal
nerve cord with different concentrations of carbachol (Research
Biochemical International, Natick, MA). To restrict the action
of carbachol to abdominal ganglia, they were separated from the
thoracic ganglia with petroleum jelly (Vaseline) dams. The volume of
this experimental chamber was <3 ml, and normal saline was
continuously perfused through the dish at 2.5-2.8 ml/min before
perfusion of carbachol solutions. The level of excitation was increased
in steps: 2, 8, 14 or 15, and 20 µM carbachol in normal saline. Each
test solution was perfused >15 min, and data were collected after >6
min perfusion of each concentration.
When >14 µM carbachol was used and the period was <0.4 s, the noise level often increased and bursts of MnT units were sometimes disrupted, so we analyzed only that range of periods in which individual MnT units could confidently be discriminated.
Description and analysis of recordings
To calculate parameters that describe the activity of the
swimmeret system, we displayed activity recorded simultaneously by each
electrode and measured the times at which each burst of impulses
started or stopped, using a digitizing tablet. From these lists of
times, we calculated the periods and durations of each burst and the
phase at which each burst began relative to the motor output of the
system (Mulloney and Hall 1987). The period of each
cycle of activity was the time from the start of one burst of impulses
to the start of the next burst. The duration of each burst was the time
from which it started to the time at which it ended.
Phase describes the point in a cycle of activity at which some event
began, for example a stimulus or a burst of impulses recorded by a
second electrode on a different nerve. To measure an event's phase, we
measured its latency, the difference between the time at which it began
and the time at which the preceding burst in the reference recording
began. We also measured the period of that cycle in the reference
recording. Phasei then was calculated as
latencyi/periodi. Normally, we used PS bursts
from the most posterior ganglion, A5, as the reference for each cycle
(Mulloney 1997). To compare the distributions of phases
recorded under different conditions and different sample sizes, we
plotted their relative cumulative frequencies. This procedure sorts the
list of measured phases from smallest to largest and calculates for
each phase the proportion of the sample smaller than or equal to it
(Zar 1984
). To calculate probabilities that phases of
bursts of impulses recorded in different ganglia occurred randomly
relative to one another, we used the Kolmogorov-Smirnov test
(Zar 1984
).
To measure the changes in period of a ganglion's output that resulted from perturbations of individual interneurons, we measured the mean period of several cycles that preceded the perturbation and the period of the cycle in which the perturbation began. We then calculated the ratio of the experimental period to the mean period; if there was no change, the ratio was 1.0.
To compare directly the durations of bursts recorded under conditions
where the motor output had different periods, we calculated the
relative durations of these bursts as duration/period (Skinner and Mulloney 1998).
The intervals between individual impulses recorded from single units were measured from data recorded with Axoscope (Axon Instruments) and displayed on the computer screen, using the measurement features of that program.
To calculate descriptive statistics and ANOVA, we used SigmaStat (Jandel).
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RESULTS |
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Axons of swimmeret interneurons occur in the MnT of each abdominal segment
MnT is a small tract of axons that rises nearly vertically from
each LN and then turns medially to cross dorsal to the LG axon, but
ventral to the median giant axon to reach the midline of the ganglion
(Skinner 1985a,b
). Whenever the swimmeret system was
active, a suction electrode placed on the surface of the LG axon at the
level of the anterior edge of the LN (Fig. 1A) recorded bursts of impulses that occurred in phase with swimmeret motor output
(Fig. 2A). These bursts of
impulses in MnT occurred in each ganglion that innervated swimmerets
used for locomotion, A2 ... A5. These impulses were not synchronous
with impulses in either N1 and so were not simply axon collaterals of
swimmeret motor neurons.
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The MnT of each ganglion in the middle of the chain, A3 and A4, contained axons that fired bursts in phase with the swimmeret motor pattern and that projected anteriorly or posteriorly to neighboring ganglia (Fig. 2A). We recorded simultaneously with three suction electrodes, one on MnT in A3 (MnT3), one on MnT in A4 (MnT4), and one on the desheathed medial-dorsal connective between A3 and A4 (Fig. 1A). Each axon that ran anteriorly from A4 fired bursts of impulses in phase with PS bursts in its own ganglion (Fig. 2, A-C). Each axon that ran posteriorly from A3 fired bursts in phase with RS bursts in its home ganglion (Fig. 2, A and D).
Intersegmental tract that contained the axons of these MnT interneurons was necessary and sufficient for normal intersegmental coordination
The axons of these interneurons ran close together in the
intersegmental connectives (Fig. 2A) in roughly the same
position in each animal. This predictability allowed us to isolate the tract that contained these axons and to test the system's responses both to cutting this tract and to cutting all of the connective except
this tract. In all these experiments, the preparation was superfused
with 5-8 µM carbachol to provide a constant level of excitation to
the system (Mulloney 1997). To assess the extent of the
lesion, each preparation was fixed after the experiment, and
transverse sections of the connective above and below the cut were
compared with sections at the cut itself.
Before any parts of the connectives were cut, the phase of bursts in PS units in A3 (PS3) relative to bursts in A5 (PS5) showed little variability (Fig. 3). When all axons in the A3-A4 connectives except the tracts that contained these axons were cut (n = 3 experiments), the variability of intersegmental phase differences did not change (Fig. 3A). The inset in Fig. 3A shows the regions of the connective that were spared in this experiment. In some experiments, the mean phase shifted after the cut, but the variability of the phases around this new mean was not greater than the variability of the control in the same experiment. These results are consistent with the idea that information carried in these tracts, probably by the MnT axons, is sufficient to maintain normal intersegmental phase differences.
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When only the dorsomedial parts of areas 76 and 78 in the A3-A4 connectives that contained axons of MnT interneurons were cut (Fig. 3B), the normal coordination of swimmeret activity in these ganglia collapsed (n = 6 experiments). After the tracts were cut, both ganglia continued to produce periodic bursts of PS impulses, but the variation in the intersegmental phase differences was much greater, and PS3 bursts drifted through the subsequent PS5 periods (data not shown). The inset in Fig. 3B shows the region of the connective that was spared in this experiment. These results are consistent with the idea that information carried in these tracts, probably in the MnT axons, is necessary to maintain normal intersegmental coordination.
If the axons in these tracts were the only ones that contributed to
intersegmental coordination, we would predict that once the tracts had
been cut the phases of PS3 relative to PSE5 would be distributed
randomly. In a cumulative frequency plot of these phases, randomly
distributed phases would lie on the diagonal line from lower left to
upper right (Zar 1984). This was not the case (Fig.
3B); a Kolmogorov-Smirnov test of the cumulative
probabilities of these measured bursts gave P < 0.001 that the phases of PS3 bursts relative to PS5 were distributed randomly
in this experiment. The classic descriptions of swimmeret coordinating
units included an ascending axon located in area 81, the lateral region
of the connective (Stein 1971
; Wiersma and Hughes
1961
). In experiments in which we cut only the medial tracts
that contained axons of MnT interneurons, this other unit would have
been spared and so might account for this departure from randomness.
Nonetheless the information carried in the surviving regions of the
connective was not enough to maintain normal coordination (Fig.
3B), and we conclude that axons in the severed regions, the
medial parts of areas 76 and 78, are necessary for normal
intersegmental coordination.
Characteristics of different types of coordinating interneurons
We have identified three types of intersegmental coordinating
interneurons that have an axon in the MnT of each abdominal ganglion
that innervates swimmerets. They originate in the clusters of cell
bodies anterior and posterior to the base of N1. Their neurites pass
through the LN before they project into the MnT and exit the ganglion
(Fig. 4). These interneurons occur as
bilateral pairs in each ganglion and can be distinguished by their
patterns of firing during swimmeret activity, by the direction of their axonal projections, by their effects on PS activity in their target ganglia, and by their structures in their home ganglia. Two types, ASCE and ASCL, send axons anteriorly from their
home ganglia, in an ascending direction (Fig. 4, A and
B). The third type, DSC, sends its axon posteriorly, in a
descending direction (Fig. 4C). Remarkably, these are
unilateral interneurons; their structures within their home ganglia are
restricted to one half of the ganglion. Although other axons do cross
through the MnT from one LN to the other (Paul and Mulloney
1986), as these coordinating axons approach the midline they
leave MnT to enter the interganglionic connective.
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Interneuron ASCE fired bursts of impulses
simultaneously with bursts in the excitatory PS motor neurons (PSE) in
its home ganglion and sent an ipsilateral axon ascending to the next
anterior ganglion (Fig. 2, A and B). We recorded
from and filled this interneuron six times. Each of these interneurons
had a small elliptical cell body, 23 × 33 µm in diameter,
located in the cluster of PSE cell bodies posterior to N1
(Sherff and Mulloney 1997). The neurite of this neuron
projected anteriorly into the lateral neuropil, where it gave rise to
several short branches as it rose dorsally in the MnT. The neurite
crossed dorsal to LG but turned before reaching the midline to project
a relatively small axon anteriorly; its structure in its home ganglion
is completely unilateral (Fig. 4).
When the system was not active (Fig.
5A), driving one
ASCE interneuron excited PSE bursts in more anterior
ganglia but had little effect on the ganglion from which it originated.
This response in the target ganglion was the same as the response
reported by Stein (1969, 1974
) to stimulation of
bundles of axons that contained a swimmeret coordinating fiber. When
the system was active, the membrane potentials of these interneurons
oscillated in phase with the motor output from their home ganglion, and
they fired bursts of impulses simultaneously with bursts in
power-stroke excitor (PSE) motor neurons in its home ganglion (Fig. 5,
D-F).
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When the system was actively producing swimmeret motor output, depolarizing currents injected into ASCE shortened the period of the next PS3 burst. Experimental hyperpolarization that inhibited firing of this ASCE decreased PS activity only slightly in A4, the neurons's home ganglion, but caused a notable decrease of PS3 activity in the neurons's target ganglion (Fig. 5C). This hyperpolarization did not affect the period of the swimmeret motor pattern, but phasic firing of this interneuron appeared to affect the excitatory drive that underlies each PS burst in the next anterior ganglion.
In a few preparations, we recorded from and filled an ascending coordinating interneuron that fired only late in the PSE burst in its home ganglion but had a posterior cell body and overall structure like ASCE neurons and had the same excitatory effect on the target ganglion that ASCE neurons do. We therefore think that these neurons with delayed bursts of impulses were nonetheless ASCE neurons. In a few preparations, we also saw physiological evidence of two pairs of ASCE neurons in one ganglion, but we observed no signs of electrical coupling of these interneurons to other cells.
Interneuron ASCL fired bursts of impulses that began late in each PSE burst in their home ganglion (Fig. 2, A and C). The numbers of impulses in the these bursts were characteristically smaller than in bursts of impulses in ASCE neurons. The conduction velocities of their impulses were higher than those of ASCE neurons (Fig. 2, B and C), and in many recordings the amplitudes of ASCL spikes were larger than those of ASCE spikes. Unlike ASCE, ASCL neurons did not always fire during each cycle when the system was only weakly excited (Fig. 6, B-D).
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ASCL had an elliptical cell body ~24 × 40 µm in diameter, located in the cluster of excitatory RS cell bodies (RSE) anterior to the base of N1 (Fig. 4). Its neurite projected posteriorly into the LN, where it joined the MnT. Its axon rose in the MnT, crossed LG, but turned anteriorly before crossing themidline. We recorded from and filled an ASCL interneuron once.
In active preparations, the membrane potentials of ASCL interneurons oscillated in phase with the motor output of their home ganglia (Fig. 6, B-D), but the trajectories of these oscillations differed from those of ASCE neurons (Fig. 5), reaching their peaks only at the end of each PSE burst. Depolarization of a individual ASCL neuron weakened PSE bursts in its home ganglion (Fig. 6A) and did not excite PSE activity in its target ganglion (Fig. 6A). After these depolarizations, PS activity in the target ganglion showed some rebound excitation (Fig. 6A).
During the experimental hyperpolarization of ASCE shown in
Fig. 5C, the ASCL unit that was also active did
not change its firing pattern at all. This lack of any effect on
ASCL implies that the ASCE and ASCL
interneurons are not electrically coupled and that ASCE is
also not electrically coupled to components of the pattern-generating
circuit that normally drive these two interneurons (Skinner and
Mulloney 1998).
Interneuron DSC fired bursts of impulses that alternated with PSE bursts in its home ganglion (Fig. 2, A and D). This interneuron's cell body lay anterior to the base of N1, in the cluster of RSE cell bodies and near the cell body of the ASCL neuron. Its neurite projected posteriorly to the LN, extended several short branches, and then rose in MnT to reach the midline and send an ipsilateral axon posteriorly to the next ganglion (Fig. 4).
In active preparations, the membrane potentials of DSC neurons
oscillated in phase with the motor output of their home ganglia. They
depolarized during each burst of impulses in RSE motor neurons and
fired bursts of impulses simultaneously with these RSE bursts. Both in
quiet and in active preparations, experimental excitation of individual
DSC neurons increased the strength of PSE firing in the neuron's
target ganglion (Fig. 7B), as
predicted by Stein (1970). Unlike ASCE,
however, hyperpolarizing DSC in active preparations did not affect PS
activity in the interneuron's target ganglion (Fig. 7C).
Furthermore, the response of the target ganglion to depolarization of
DSC in quiet preparations was relatively slow (Fig. 7A).
These differences suggest that ASCE and DSC have
different synaptic targets in each module (cf. Skinner
and Mulloney 1998
).
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In one preparation, we observed two sizes of DSC impulses (Fig.
7C), which suggests that there might be two pairs of these interneurons per ganglion, consistent with Stein's
(1971) description of units in the medial descending tract.
Firing of these interneurons was coordinated with the motor output from their home ganglia
These three types usually fired bursts of impulses when the system
was producing the normal swimmeret motor pattern (Fig. 2). These bursts
occurred at a predictable point in each cycle of the motor pattern
(Fig. 8, and probably reflect a strong
synaptic drive that is common to these interneurons and to the motor
neurons that fire simultaneously with them (Mulloney et al.
1997; Skinner and Mulloney 1998
).
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Responses of the swimmeret system to perturbation of individual coordinating interneurons
If these interneurons carry the information needed to coordinate activities of swimmeret modules in different segments, then disrupting the firing of individual interneurons should affect the intersegmental phase difference unless the system was constrained too tightly by the continued activity of other coordinating interneurons. We stimulated individual ASCE and DSC interneurons to test the responses of their respective target modules. These stimuli began at different phases in the PS firing cycle of the target ganglion (Fig. 9).
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The responses to stimulation of individual ASCE neurons were contingent on the phase in the target ganglion's cycle at which the stimulus occurred. If the stimulus began while PSE units in the target were firing, the next PS burst in the target ganglion was delayed. If the stimulus began while PSE units in the target were silent, the next PS burst was advanced (Fig. 9).
The responses to stimulation of an individual DSC were also contingent on the phase at which the stimulus occurred. If the stimulus began during a PSE burst in the target ganglion, the response of the target was unpredictable. If the stimulus began while the target PS units were silent, the next PS burst was advanced (Fig. 9). From these results, it is evident that each swimmeret module includes both ascending and descending interneurons that carry information about activity in their homemodule and can affect the timing of activity in neighboring modules.
In recent simulations of the coordinating circuit (Skinner and
Mulloney 1998), we have assumed that coordinating interneurons played no roles in generating the motor pattern within their home module. If this assumption was correct, then experimental stimulation of a coordinating neuron would have a greater effect on its target ganglion than on its home ganglion, although the existence of a
polysynaptic intersegmental loop might obscure this difference. To test
this assumption, we compared the responses of target modules and home
modules to the same stimuli, ignoring the phase difference between the
two. Changes in the periods of target modules in response to
stimulation of ASCE neurons were not significantly
different from the changes in their home modules (P = 0.44, n = 16 stimuli, paired t-test).
Changes in the periods of target modules in response to stimulating DSC
neurons were significantly larger than the changes in their home
modules (P = 0.016, n = 6 stimuli,
paired t-test).
Responses of coordinating interneurons to changes in excitation
When excitation of the swimmeret system increases, the frequency
of PSE and RSE bursts increase, each motor neuron fires more spikes per
burst, and previously silent motor neurons are recruited (Braun
and Mulloney 1993; Mulloney 1997
;
Mulloney et al. 1997
). Each of these effects would
contribute to more frequent and more powerful swimmeret beating, more
thrust, and so faster forward swimming, consistent with the behavior of
intact crayfish. Nonetheless the structure of the swimmeret motor
pattern is preserved; neither the phases of PSE and RSE in each segment
nor the phases of bursts in neighboring segments change significantly,
and the ratio of each burst's duration to the period of the pattern is
preserved (Skinner and Mulloney 1998
). If it is correct
that MnT units are necessary components of the intersegmental
coordinating circuit, then their responses to changes in excitation are
key features of the system's ability to preserve the structure of the
swimmeret motor pattern despite changes in period.
To study the responses of these interneurons to changes in excitation,
we recorded MnT units in A3 and A4 while varying the level of
excitation using different concentrations of carbachol, which excites
the swimmeret system in a dose-dependent manner (Mulloney
1997). We compared the mean spike frequencies, the numbers of
impulses in each burst, and the structures of individual bursts of each
unit in MnT3 and MnT4 at different periods. In preparations that were
spontaneously active in normal saline, the period of the swimmeret
motor pattern ranged from 0.6 to 0.9 s. In response to perfusion
with increasing concentrations of carbachol, the period decreased
smoothly down to ~0.35 s.
The relative durations and phases of bursts of impulses in each MnT
interneuron produced when the preparation was excited were similar to
those produced spontaneously by these same preparations (Fig.
10). In this figure, we scaled the
periods, latencies, and relative burst-durations of spontaneous
activity to the period of the excited activity (0.4 s) so that the
structures of the motor patterns could be compared directly
(Mulloney 1997). In the output from excited
preparations, there was a slight shift in the phases of bursts in
ascending axons that correlated with a similar shift in the phases of
PS3 bursts. The mean phases of ASCE changed from 0.02 ± 0.09 to 0.11 ± 0.09 and those of ASCL changed from
0.36 ± 0.05 to 0.40 ± 0.06. In the preparations used for
this figure, the relative durations of PS3 bursts did increase when the
system was excited (P = 0.03).
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We looked more closely at the structures of activity recorded at four different periods. An ANOVA in phase and relative duration of bursts of impulses in each type of coordinating interneuron revealed no significant differences in these parameters as the period of the motor pattern ranged from 0.35 to 0.75 s (for phases and relative durations of ASCE: P = 0.41, P = 0.31; of ASCL: P = 0.79, P = 0.60; of DSC: P = 0.26, P = 0.76), although the increase in relative duration of the PS3 bursts was confirmed.
Firing patterns within individual bursts of impulses
To describe the dynamics of bursts of impulses in these interneurons, we measured the time intervals between impulses within bursts in a representative series of bursts for each type of coordinating interneuron. We then plotted impulse frequencies (the reciprocals of these intervals) as functions of the order in which each impulse occurred in the burst (Fig. 11).
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During spontaneous swimmeret activity, impulse frequency in all three types of coordinating interneurons usually decreased monotonically as each burst of impulses progressed, as if the neurons were accommodating to a constant current (Fig. 11). Increased excitation changed the frequency and duration of these bursts (e.g., Fig. 6), but the impulse pattern within each burst did not normally change; the initial impulse frequency was higher but the monotonic accommodation of ASCL neurons and DSC neurons did not change (Fig. 11).
ASCE neurons responded more complexly; when the system was excited with carbachol, their impulse frequencies at first increased and then decelerated smoothly until the end of the burst (Fig. 5, C-E). Two of the six ASCE neurons studied in A3, which we encountered in preparations that were spontaneously expressing stable swimmeret motor patterns, also fired bursts that had an initial acceleration before these preparations were exposed to carbachol.
Mean impulse frequency of each MnT coordinating interneuron increased, but the numbers of impulses per burst did not change
The mean impulse frequency of each coordinating interneuron recorded was measured for four different ranges of periods (Fig. 12A). Each point plotted in this figure is the mean impulse frequency of that MnT neuron for all experiments within that range. As period decreased, the firing frequency of each unit increased as would be expected in response to excitation.
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However, because the duration of each burst decreased proportionately to the decrease in period, the numbers of impulses in each burst did not change significantly (Fig. 12B). An ANOVA found it probable that the mean numbers of impulses were not different in this range of periods (ASCE: P = 0.915, ASCL: P = 0.658, DSC: P = 0.299). This feature is not a necessary consequence of the preservation of relative burst duration (Fig. 10) but instead is an unexpected property of these interneurons.
From this evidence, we think that all the coordinating interneurons are
affected in the same way by excitation of the swimmeret system: they
receive stronger synaptic excitation and therefore fire at higher
frequencies, but they do not change their firing properties. Like
swimmeret motor neurons (Sherff and Mulloney 1996), the
periods, durations, and phases of their bursts of impulses are
determined largely by the synaptic input they get from the pattern-generating interneurons within their home ganglion.
Each abdominal ganglion had the same complement of coordinating axons in MnT
To discover the structures of these MnT neurons, we used Neurobiotin to backfill bundles of axons stripped from the dorsomedial face of each hemiconnective (areas 76 and 78; cf. insets in Fig. 3) between A3, A4, and A5 and studied the filled neurons as whole mounts (e.g., Fig. 1B). The numbers of ascending axons and descending axons filled in MnT was similar in A2, A3, A4, and A5 (Table 1). At least two interneurons the axons of which ran in MnT were filled in each preparation made from anterior to the ganglion. At least two other interneurons with axons in MnT were also visible in each preparation made from posterior to A2 and A3. Fills from the posterior direction also revealed that MnT sometimes divided laterally before crossing LG. In these preparations, ascending axons crossed LG near the anterior edge of LN but descending units crossed LG near the neuropil's posterior edge, so that anterior-projecting units were separated from posterior-projecting units in our standard recording position (Fig. 1A). We interpret this variation as individual differences in the local position of a divergence that is present in the MnT of every ganglion.
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We compared the numbers of units recorded by an extracellular MnT electrode, positioned at the anterior edge of LN (Fig. 1A), with the numbers of backfilled neurons in ganglia A2, A3, A4, and A5, the ganglia that in this species innervate functional swimmerets (Table 1). The numbers of filled axons in MnT were similar in each ganglion, but the numbers of units recorded that fired in phase with swimmeret activity were more variable. This disparity might mean that there are segmental differences in the numbers of ascending and descending coordinating axons, or it might be caused simply by mechanical damage from removing the sheath or placing the electrode because the axons in MnT are quite fine (Fig. 1C). We think there is another cause. When recording on the surface of LG at the posterior edge of LN in some preparations, we found units that fired in phase with the swimmeret system. This is the position where backfills had revealed that axons with the structure of coordinating interneurons sometimes crossed LG before joining the rest of MnT. In recordings from this more posterior position in A3, we found at least two units that fired simultaneously with RSE motor axons. Simultaneous recordings from the three to four connective showed that, like DSC interneurons, these axons also projected posteriorly in the dorsal-medial connective. In A4, we did not find descending units while recording from the standard MnT position, but more posterior recording positions in the same preparations found one or two descending units. These differences in recording position and routes of filled axons suggest that axons of ascending and descending interneurons do rise together toward the lateral edge of LG but can diverge even before turning toward the midline and that this divergence is more probable in posterior ganglia than in anterior ones. If this inference is correct, then the disparities in the numbers of active swimmeret units recorded in different ganglia (Table 1) might be due to this segmental difference in the course of MnT not to a segmental difference in the numbers of coordinating interneurons. Although our evidence is incomplete, we think that each ganglion that innervates swimmerets has the same complement of MnT coordinating interneurons.
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DISCUSSION |
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The three types of MnT interneurons described here differ from
other swimmeret neurons in their physiology and anatomy (Paul and Mulloney 1985b, 1986
; Sherff and Mulloney
1997
). Unlike command interneurons, the axons of which project
the length of the abdominal nerve cord (Acevedo et al.
1994
), these interneurons occur as bilateral pairs in each
intermediate ganglion that innervates swimmerets and send axons into
the intersegmental connective that project to neighboring ganglia (Fig.
4). Firing of these MnT units affected the timing of the next cycle of
motor output from their target ganglia (Fig. 9), a property
characteristic of coordinating interneurons (Paul and Mulloney
1986
; Stein 1971
, 1974
). Each type of MnT
coordinating interneuron fired bursts of impulses at a different phase
of the swimmeret cycle in its home ganglion (Fig. 8), so we infer that
these bursts convey different information to their targets in
neighboring ganglia.
MnT interneurons are both necessary and sufficient for normal coordination of swimmeret motor patterns
Intersegmental coordination requires cycle-by-cycle information
about the state of each swimmeret, and only neurons that fire impulses
in phase with the local circuits driving individual swimmerets can
conduct the necessary information. Each half of the bilaterally symmetrical connective that runs between neighboring abdominal ganglia
contains ~2,600 axons (Sutherland and Nunnemacher
1968). Of these 2,600, only three "tracts" have been
reported to contain axons that fire bursts of impulses in phase with
the swimmeret motor output from their home ganglion (Skinner and
Mulloney 1998
; Stein 1971
; Wiersma and
Hughes 1961
). Of these three, one ascending tract and one
descending tract run in the dorsomedial region of the connective; the
third runs near the lateral edge of the connective (cf. Fig.
2A).
We could sever selectively either the medial ascending and descending
tracts or the rest of the connectives including the lateral ascending
tract (Fig. 3). Cutting all the intersegmental axons except those in
the dorsomedial tracts did not affect coordination, so we conclude that
the ascending units in the lateral ascending tract are not necessary
for normal coordination, and those units in the medial tracts are
sufficient for normal coordination. The reciprocal experiment, cutting
only the dorsomedial tracts, uncoupled activity in ganglia on opposite
sides of the cut (Fig. 3B), so we conclude that the
ascending axons in the lateral tract are not sufficient for normal
coordination, and those units in the medial tracts are necessary for
normal coordination (cf. Figs. 3, 4 of Stein 1971).
Not all three types of MnT coordinating interneuron reached threshold whenever the swimmeret system was active. ASCE fired periodic bursts whenever the system began to oscillate, and we have argued above that the absence of DSC bursts from some MnT recordings was caused by individual differences in the courses through MnT followed by ascending and descending axons. ASCL, however, sometimes failed to reach threshold (e.g., Fig. 6, B and C), although the intersegmental coordination of preparations in which we observed this was apparently normal. Thus although the flow of information through MnT interneurons is essential for normal coordination (Fig. 3), participation by every one of the MnT interneurons in each segment is not necessary.
These interneurons are components of a concatenated intersegmental coordinating circuit
As they project axons from their home ganglia to their targets in neighboring ganglia, they form a concatenated pathway through which information about the state of each local swimmeret module is exported to its neighbors. Although we assessed the impact of impulses in these interneurons on their targets by measuring changes in the timing of bursts in PS motor neurons, it is unlikely that the major targets of ASCE or DSC are the PS motor neurons themselves because the responses of the target modules were contingent on the phase at which these interneurons were stimulated (Fig. 9). This phase-dependent response of the target, which we observed both for ascending and for descending MnT interneurons, means that these interneurons can effectively hold the target module to a particular phase relative to their home module's activity.
Experiments with nonuniform excitation of the system (Braun and
Mulloney 1995) predict that the structure of this coordinating circuit is asymmetric. Simulations of the swimmeret system as a chain
of coupled oscillators (Skinner et al. 1997a
,b
) showed that only a restricted set of properties of the information linking neighboring oscillators permitted the simulated system to reproduce the
performance of the real system when it was not uniformly excited (Braun and Mulloney 1995
). These restrictions included
that the ascending and descending coupling were asymmetric and that
changes in coupling did not have much effect on the period of the
system's output. Those properties of MnT coordinating interneurons
that we have investigated thus far conform to these specifications. ASCE and ASCL fire during the PS part of the
cycle, but DSC fires during the RS part (Fig. 8), so there is an
asymmetry in the timing of the ascending and descending signals from
each ganglion.
These MnT interneurons are the classically described swimmeret coordinating fibers
The original reports of swimmeret coordinating fibers
(Stein 1971) described two units in the median ascending
tract (area 78) of the connectives between each ganglion. These
ascending units fired bursts of impulses that propagated anteriorly, in an "ascending" direction; a small unit that fired simultaneously with each burst of impulses in PS motor neurons and a large unit that
fired late in each PS burst. Stein (1974)
showed that
stimulating a burst of impulses in these medial ascending units would
either advance or retard the next PS burst in the target ganglion,
depending on the phase at which the firing of the ascending units
began. This is precisely the result shown in Fig. 9, here demonstrated by intracellular stimulation of single interneurons. Two more units in
the medial descending tract (area 76, near area 78) fired bursts that
propagated posteriorly, in a "descending" direction, during each
burst of impulses in RS motor neurons.
The positions of these MnT units in areas 76 and 78 of the
interganglionic connective (Wiersma and Hughes 1961),
the phases in the swimmeret cycle at which they normally fire (Figs.
2A and 8), and their effects on the modules to which they
projected (Fig. 9) match those of coordinating fibers classically
described. We think that axons of ASCE and ASCL
interneurons are homologous to Stein's coordinating fibers in the
medial ascending tract, and axons of DSC interneurons are homologous to
his fibers in the medial descending tract.
Dynamics of MnT coordinating interneurons
Each swimmeret module can respond to pharmacological excitation
applied locally (Acevedo et al. 1994; Braun and
Mulloney 1995
), and each is capable of oscillating
independently (Murchison et al. 1993
). Changes in
excitation affect the period of the swimmeret motor pattern but do not
affect either the phases of PS-RS alternation within each ganglion or
the intersegmental phase lags between neighboring ganglia (Braun
and Mulloney 1995
; Mulloney 1997
; Skinner and Mulloney 1998
). In these experiments as we increased
excitation, the period of the motor pattern shortened, but neither the
phases nor the relative durations of bursts of impulses in the MnT
interneurons changed (Fig. 10). This feature of their responses to
excitation resembles the responses of swimmeret motor neurons, which
also preserve relative duration as period changes (Mulloney
1997
; Skinner and Mulloney 1998
).
Excitation increased the frequency and amplitude of synaptically driven
oscillations of membrane potential in MnT interneurons (Figs. 5 and 6)
(cf. Mulloney et al. 1997) and causes higher impulse frequencies within each burst (Fig. 11). The increased firing frequency would produce stronger synaptic input to the target ganglion during each cycle and increase coupling between each module. This increase may
contribute to the stability of intersegmental phase differences.
One unpredicted feature these interneurons' responses to increased
excitation was that the mean numbers of impulses per burst remained
unchanged through a wide range of periods (Fig. 12). For each type of
MnT interneuron, the increased impulse frequency was matched with the
decreased burst duration so that the mean number of impulses per burst
did not vary significantly in the range of periods we examined. This
preservation of impulse number is not a necessary consequence of
preserving relative burst duration. Instead it might reflect a feature
of the nonspiking interneurons in each local pattern-generating circuit
that are thought to provide the excitatory synaptic drive to the MnT
interneurons (Skinner and Mulloney 1998). The
postsynaptic consequences of preserving spike number as period changes
have been studied in stomatogastric muscles (Morris and Hooper
1997
), but here the consequences must await the identification
of neurons postsynaptic to MnT interneurons in their target ganglia.
Intersegmental coordination in other locomotor systems
Locomotion in other segmented animals also is coordinated by
intersegmental interneurons. In walking insects, intersegmental interneurons fire bursts of impulses in phase with the step cycles in
their home ganglion (Pearson and Iles 1973) and
intersegmental interneurons that are probably active during walking
coordinate limb movements during grooming (Berkowitz and Laurent
1996
). We suspect that these insect interneurons will prove to
be homologues of crustacean coordinating interneurons, but the evidence
is too incomplete to decide the question.
In walking reptiles and mammals, propriospinal interneurons perform the
same functions. In turtles, a heterogeneous population of descending
propriospinal interneurons fire bursts of impulses during rhythmic
scratching movements (Berkowitz and Stein 1994). Some
project ipsilaterally, others contralaterally (Berkowitz and
Stein 1994
). These interneurons drive segmental circuits that generate motor patterns for directed scratching movements (Stein et al. 1995
).
In animals that swim by undulations of a segmented trunk, there are
also intersegmental coordinating interneurons essential for normal
behavior. Leeches have ventrally positioned segmental ganglia that
innervate their swimming musculature. The activity of the
pattern-generating circuit in each ganglion is coordinated by a
concatenated intersegmental circuit the component interneurons of which
occur in a segmental series like the MnT interneurons, but the axons of
which project beyond their neighboring ganglia (Friesen and
Pearce 1993). Within their target ganglia, these coordinating
interneurons synapse directly with local interneurons in the segmental
pattern-generating circuit. This swimming circuit in leeches has many
properties similar to the swimmeret system.
Lamprey and other fish have spinal pattern-generating circuits that are
coordinated by intersegmental axons, but in these animals the roles of
different interneurons is less clear. The kernel of each segmental
pattern-generating circuit includes interneurons that both participate
in the segmental pattern-generating circuit (Grillner and
Matsushima 1991) and contribute to intersegmental coordination
(Buchanan 1992
; Williams 1992
). Moreover
in lamprey the segmental boundaries apparent in the motor output are
obscure or not present in the premotor pattern-generating circuitry
(Lansner et al. 1997
), which makes it difficult to
define the synaptic connections within and between neighboring
circuits. Nonetheless coupling between nearby segments is essential for
the normal phase-constant performance of the swimming circuit.
Directions for further investigation
We do not yet know the neuronal targets of these MnT interneurons
in the ganglia to which they project nor the sources of the synaptic
currents that make them fire when the system is active. Simulations of
alternative intersegmental coordinating circuits, using a recently
developed cellular model that assumes a particular organization of each
local module (Mulloney et al. 1998; Skinner and
Mulloney 1998
), have predicted a particular configuration of
targets and synapses that are consistent with what we know thus far
about the MnT interneurons. By mapping the synaptic connections of MnT
interneurons in their home ganglia and their target ganglia, we hope to
reveal more of the cellular basis of this complex behavior.
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ACKNOWLEDGMENTS |
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We thank W. M. Hall for data analysis, editorial criticism, and preparing illustrations. This work was supported by National Science Foundation Grants IBN 95-14889 and IBN 97-28791 to B. Mulloney.
Present address of H. Namba: Dept. of Molecular Neurobiology, Brain Research Institute, Niigata University, Niigata 951-8122, Japan.
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FOOTNOTES |
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Address for reprint requests: B. Mulloney, Neurobiology, Physiology, and Behavior, University of California, Davis, 1 Shields Dr., Davis, CA 95616-8519.
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 18 December 1998; accepted in final form 1 February 1999.
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REFERENCES |
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