First published online March 21, 2005
Journal of Experimental Biology 208, 1347-1361 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01500
Central pattern generator for swimming in Melibe
Stuart Thompson1,* and
Winsor H. Watson, III2
1 Department of Biological Sciences, Hopkins Marine Station, Stanford
University, Pacific Grove, CA 93950, USA
2 Zoology Department, Center for Marine Biology, University of New
Hampshire, Durham, NH 03824, USA
*
Author for correspondence (e-mail:
stuartt{at}stanford.edu)
Accepted 18 January 2005
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Summary
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The nudibranch mollusc Melibe leonina swims by bending from side
to side. We have identified a network of neurons that appears to constitute
the central pattern generator (CPG) for this locomotor behavior, one of only a
few such networks to be described in cellular detail. The network consists of
two pairs of interneurons, termed `swim interneuron 1' (sint1) and
`swim interneuron 2' (sint2), arranged around a plane of bilateral
symmetry. Interneurons on one side of the brain, which includes the paired
cerebral, pleural and pedal ganglia, coordinate bending movements toward the
same side and communicate via non-rectifying electrical synapses.
Interneurons on opposite sides of the brain coordinate antagonistic movements
and communicate over mutually inhibitory synaptic pathways. Several criteria
were used to identify members of the swim CPG, the most important being the
ability to shift the phase of swimming behavior in a quantitative fashion by
briefly altering the firing pattern of an individual neuron. Strong
depolarization of any of the interneurons produces an ipsilateral swimming
movement during which the several components of the motor act occur in
sequence. Strong hyperpolarization causes swimming to stop and leaves the
animal contracted to the opposite side for the duration of the
hyperpolarization. The four swim interneurons make appropriate synaptic
connections with motoneurons, exciting synergists and inhibiting antagonists.
Finally, these are the only neurons that were found to have this set of
properties in spite of concerted efforts to sample widely in the
Melibe CNS. This led us to conclude that these four cells constitute
the CPG for swimming. While sint1 and sint2 work together
during swimming, they play different roles in the generation of other
behaviors. Sint1 is normally silent when the animal is crawling on a
surface but it depolarizes and begins to fire in strong bursts once the foot
is dislodged and the animal begins to swim. Sint2 also fires in
bursts during swimming, but it is not silent in non-swimming animals. Instead
activity in sint2 is correlated with turning movements as the animal
crawls on a surface. This suggests that the Melibe motor system is
organized in a hierarchy and that the alternating movements characteristic of
swimming emerge when activity in sint1 and sint2 is bound
together.
Key words: pattern generator, locomotor system, nudibranch, Melibe leonine
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Introduction
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The orchestration of animal locomotion involves networks of neurons in the
central nervous system that function as the central pattern generator (CPG)
for the behavior and are responsible for coordinating locomotor movements
(Stent et al., 1978
;
Delcomyn, 1980;
Grillner and Wallen, 1985;
Grillner et al., 1989;
Getting, 1988,
1989b). While this concept is
widely accepted (Friesen,
1994), few examples of locomotor CPG networks are known in detail.
The nudibranch mollusc Melibe leonina swims by bending from side to
side in a behavior that can continue for hours in freely swimming animals
(Hurst, 1968; Watson et al.,
2001,
2002;
Lawrence and Watson, 2002). We
studied the neural network responsible for generating the swimming rhythm in
Melibe using microelectrode techniques applied to whole animal
preparations, in which cellular activity and behavior could be recorded
simultaneously. We also studied specific features of the Melibe swim
CPG in isolated ganglion preparations that continue to express the swimming
motor program ex vivo. Using these two approaches, we were able to
identify a network of interneurons in the central nervous system that is
responsible for determining the form, frequency and amplitude of swimming
movements. We believe this network represents the core and possibly the
entirety of the central pattern generator for Melibe swimming.
We propose that the swim CPG consists of two pairs of interneurons.
Interneurons on the same side of the brain function as synergists and are
electrically coupled, while interneurons on opposite sides of the brain are
antagonists and communicate over mutually inhibitory synaptic pathways. Mutual
inhibition appears to be critically important for the expression of
alternating activity in the network. Variations on this theme appear again and
again in the analysis of oscillatory networks in central nervous system
structures at every level of complexity, from molluscan ganglia to mammalian
cortex. It is one of the core motifs in neuronal architecture. In concept, the
Melibe swim CPG resembles the paired half-centers model
introduced by Graham Brown
(1911) that provided an early
model for the stepping pattern generator in mammalian locomotion. The relative
simplicity of the Melibe system has allowed us to study some of the
properties of half-center networks in physiological rather than
computational experiments. The interneurons thought to constitute the central
pattern generator for swimming, their synaptic interactions, and their output
to motoneurons are described here.
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Materials and methods
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Specimens of Melibe leonina Gould were collected near the Hopkins
Marine Station, Pacific Grove, CA, USA and near Friday Harbor Laboratories,
Friday Harbor, WA, USA and kept in flowing seawater aquaria at ambient
temperature (
15°C; Schivell et
al., 1997). Most electrophysiological recordings were made using
the whole animal preparation developed by Dennis Willows and colleagues
(Dorsett et al., 1969;
Getting, 1989a;
Willows, 1991). This report is
based on the results of 73 successful whole-animal experiments in which the
animal was able to swim normally throughout the period of intracellular
recording.
The brain, which consists of the fused cerebral, pleural and pedal ganglia,
was exposed by a small dorsal incision and stabilized against a rigid platform
while the animal was suspended in a tank of cooled re-circulating seawater at
15°C (Watson et al.,
2002). Melibe can be induced to swim when prepared in
this way by depriving the animal of a surface to stand on. Swimming movements
were monitored by tying a suture through the posterior tip of the body and
connecting it to a lever that partially shielded a photocell. This device
produces an oscillating output during swimming with maximum voltage at the
peak of the movement to the right and minimum voltage at the peak of the
movement to the left. It allowed us to monitor the timing of swimming
movements, but occasionally the detector restricted the movement and did not
accurately record the maximum excursion.
Melibe neurons are not distinctly pigmented
(Cohen et al., 1991) and their
locations are somewhat variable between preparations. We used the whole animal
preparation in most of our experiments, which allowed individual neurons to be
tested for functional equivalence on the basis of the movement produced when
stimulated, the phase relationship between activity and behavior, the pattern
of synaptic input received during swimming and during rest, and the response
to tactile stimulation. These criteria for the identification of equivalent
neurons were applied in every experiment. Intracellular recordings were made
from neuron cell bodies using glass microelectrodes filled with 3 mol
l–1 KCl (Re=20–40 M
).
Stimulating currents were applied via the recording electrode using a
constant current source and a bridge-circuit to null the voltage drop across
the electrode resistance. The motor program for swimming continues to be
expressed in the Melibe central nervous system after cutting all of
the nerve trunks exiting the brain, taking care to leave the circumesophageal
connectives intact (Watson et al.,
2002). We refer to expression of the motor program in the isolated
CNS as fictive swimming.
The isolated nervous system was removed to a Plexiglass chamber and bathed
in filtered natural seawater or in physiological saline containing (mmol
l–1); 470 NaCl, 10 KCl, 10 CaCl2, 50
MgCl2, 10 Hepes (pH 8) at 15°C. Data from five isolated nervous
system preparations contributed to this report. In order to view the axonal
projections of interneurons, cell bodies was injected with Lucifer Yellow (LY)
by electrophoresis from an intracellular electrode containing a 5% solution of
LY in 0.15 mol l–1 LiCl (N=24) using 500 ms, 10 nA
current pulses applied at 1 Hz for 20 min. Preparations were fixed in 4%
paraformaldehyde overnight and viewed using an epifluorescence microscope.
Where appropriate, values in the text are specified as mean ±
S.D.
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Results
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Melibe swimming results from alternating activity in two
motoneuron pools responsible for producing left and right bending of the body
(Watson et al., 2002).
Changing the firing pattern of single motoneurons or pairs of motoneurons does
not change the frequency or phase of swimming, indicating that the motoneurons
are not part of the central pattern generator for the behavior and do not
directly influence pattern generation (S. H. Thompson, unpublished
observations). Instead, motoneurons appear to function as a common path for
behaviors that involve the same sets of body wall musculature. The question
that arises is what binds multipurpose motoneurons together in order to
generate the coordinated movements involved in swimming? Watson et al.
(2002) showed that motoneurons
that function as synergists during swimming fire in-phase because of shared
excitatory synaptic drive, while those that function as antagonists fire
out-of-phase because of alternating drive. The convergent synaptic drive onto
motoneurons suggests that there must be pre-motor neurons that feed-forward to
excite synergists and inhibit antagonists. A search for the sources of
synaptic drive onto motoneurons during swimming led to the identification of
two important classes of central nervous system interneurons.
One interneuron in each of the two pleural ganglia fires strong bursts of
action potentials in-phase with swimming and makes synaptic connections with
motoneurons that drive the movement. These two cells have been named the right
and left `swim interneuron 1' (Rsint1 and Lsint1; referred
to as SiI by Watson et al.,
2001). Their cell bodies are located on the medial dorsal surface
just caudal to the prominent tentacular lobe that rises from the center of the
pleural ganglion (Fig. 1). The
cell body of sint1 is unpigmented and 30–50 µm in diameter.
It is surrounded by similar looking cells but can be identified on functional
grounds because it is the only neuron in the region that fires bursts of
action potentials phase-locked to swimming movements. The axon distribution of
sint1 was examined after injecting LY into the soma
(Fig. 1). Sint1
branches in the pleural ganglion neuropil near the base of the optic lobe and
projects to the ipsilateral pedal ganglion via the dorsal
pleural-pedal connective, where it forms a series of arborizations. We did not
observe processes projecting to the contralateral side via the
central commissure or circumesophageal connectives and no processes were seen
to exit the central nervous system.
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Fig. 1. Axon distributions of sint1 and sint2. (Top) A diagram of
the dorsal aspect of the Melibe (rostral at the top), illustrating
the three major subdivisions of the CNS: the cerebral ganglia (C), pleural
ganglia (Pl) and pedal ganglia (Pd). The cell bodies and major projections of
sint1 and sint2 are shown diagrammatically, based on
interpretation of 24 successful Lucifer Yellow dye fills. Examples of Lucifer
Yellow stained interneurons are shown below. Sint1 branches near its
cell body located just caudal to the tentacular lobe (T) in the pleural
ganglion and sends a process to the ipsilateral pedal ganglion that arborizes
in the pedal ganglion neuropil. It does not project across the midline
via the central commissure or beyond the CNS. Sint2 branches
in the pedal ganglion and sends a process via the pedal-pedal
connective to the opposite pedal ganglion. No processes were seen to project
to the pleural ganglia or leave the CNS. The dye fills illustrated in this
figure were done in isolated ganglion preparations.
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A second pair of interneurons with cell bodies located in the pedal ganglia
shares many of the properties of sint1. These two cells are termed
the right and left `swim interneuron 2' (Rsint2 and Lsint2;
referred to as SiII by Watson et al.,
2001). A single sint2 is found near the dorsal midline of
each pedal ganglion (Fig. 1).
Like the motoneurons in the pedal ganglia
(Watson et al., 2002),
sint2 fires strong bursts of action potentials during swimming but
can be easily distinguished from motoneurons on the basis of the synaptic
connections it makes with sint1, its influence on the timing of
swimming movements, and its synaptic output to motoneurons. LY staining shows
that sint2 branches in the pedal ganglion neuropil and sends a major
process to the opposite pedal ganglion via the circumesophageal,
pedal-pedal connective. No processes were seen to travel directly to the
pleural ganglia or exit the central nervous system. The detailed properties of
sint1 and sint2 are described below.
Swim interneuron 1 (sint1)
The pattern of activity in Rsint1 during a brief episode of
swimming is shown in Fig. 2A
along with a record of swimming behavior. Swimming was initiated by removing a
surface from the animal's foot at the first arrow (movement at this time is
due to the physical intervention) and terminated by returning the surface at
the second arrow. Sint1 is silent in non-swimming animals and rarely
fires even in response to tactile stimulation. Once the animal is dislodged,
Rsint1 immediately depolarizes and begins to fire in a bursting mode
coincident with the beginning of swimming. In contrast to spike bursts in
motoneurons (Watson et al.,
2002), the bursts in sint1 ride on a depolarized plateau
such that the membrane voltage during the intervals between bursts is
5–10 mV more positive than the voltage recorded when the animal is
quiescent or crawling on a surface. The implication is that sint1 is
tonically inhibited when the foot is in contact with a surface, but once
contact is broken and inhibition is removed, sint1 depolarizes to a
level sufficient to maintain spiking activity.
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Fig. 2. Firing pattern in swim interneuron 1. (A) Lower trace: intracellular
recording from Rsint1 during a short episode of swimming. Swimming
was initiated by separating the animal's foot from a surface at the first
arrow and terminated by returning the surface at the second arrow. Upper
trace: a record of the animal's side-to-side swimming movements (upward
deflection indicates bending toward the right). (B) Lower trace: bursting
activity in Rsint1 at higher gain and on an expanded time base.
Insert: the trajectory of membrane voltage at the end of a burst on an
expanded scale. Action potentials were truncated by the recording device in
this example. Upper trace: a record of the animal's movement. (C) Simultaneous
recording of activity in Rsint1 (middle) and Lsint1 (bottom)
during a long swimming episode along with the record of swimming movements
(top). Note that the spike bursts in these two cells do not overlap.
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The action potential burst in sint1 precedes the movement to the
ipsilateral side and occupies about 40% (39.1±6.2%; N=27) of
the swim period. It begins before the movement toward the opposite side
reaches its peak and continues into the beginning of the movement to the same
side, ending before the peak of the ipsilateral movement. Using the time of
maximum ipsilateral bending as a reference, the burst begins
250.1±24.7° (N=27) before the peak and ends
114.2±28.6° (N=27) before the peak. In each individual
preparation, the phase relationship is maintained throughout long episodes of
swimming but the number of action potentials in the burst (the burst size) can
be more variable. In one example, burst size varied between 11 and 33 spikes
per burst over 50 cycles of continuous swimming. Variability in burst size in
sint1 may explain the observation that the amplitudes of swimming
movements wax and wane during long bouts of swimming while the frequency
remains more constant. Swimming ends abruptly when the foot contacts a surface
(Fig. 2A). As contact is made,
sint1 is immediately hyperpolarized and becomes silent, again
suggesting that sint1 may receive tonic inhibition as a direct result
of foot contact. The bursting activity in Rsint1 during swimming is
shown at higher gain and on an expanded time scale in
Fig. 2B. This record shows that
action potential bursts in sint1 are shaped by strong synaptic input.
It appears that burst termination results from both cessation of excitatory
drive and the appearance of ipsp input that continues into the interburst
interval (see insert). Fig. 2C
shows a simultaneous recording of activity in Rsint1 and
Lsint1 during swimming. The two antagonistic interneurons fire in
antiphase and there is no overlap between the bursts in these two cells. The
interval between the last spike in the burst in one neuron and the first spike
in its homologue is fairly constant (519±60 ms; N=20). This is
a defining feature of the swim CPG and suggests that the network includes
mechanisms for maintaining a nearly constant latency between bursts in the two
sint1 values.
Influence of sint1 on behavior
Stimulation of sint1 to fire a burst of action potentials in a
quiescent animal causes a bending movement toward the ipsilateral side and the
animal remains in that posture for the duration of the stimulus. The driven
movement appears to include all of the components that occur during swimming
and closely resembles the swimming movement. Lawrence and Watson
(2002) described
Melibe swimming in detail. Swimming movements result from
contractions of the longitudinal and diagonal body wall musculature.
Contraction of longitudinal muscles pulls the anterior and posterior of the
animal together to form a C-shaped bend. Muscles that course dorsally over the
oral hood pull the dorsum of the hood to the contracting side and this imparts
a corkscrew twist to the body. Stimulation of a single sint1 results
in the same pattern of contractions. Similarly, when sint1 is
stimulated to fire a prolonged burst in a swimming animal, side-to-side
movements cease and the animal remains maximally contracted to the stimulated
side. Fig. 3 shows the effect
of applying hyperpolarizing currents of increasing strength to a single
sint1 during swimming. As the current is increased, swimming becomes
progressively disorganized. Small currents cause the period of the oscillation
to increase and cause asymmetrical contractions
(Fig. 3A–C). Strong
currents sufficient to prevent firing in the soma and axon branches of
sint1 (judged by the absence of axon spikes) interrupt swimming
altogether and cause the animal to remain contracted to the opposite side
throughout the hyperpolarization (Fig.
3D). It is particularly interesting that as the hyperpolarizing
current is increased, the phasic synaptic input normally seen in the
interneuron during swimming becomes progressively diminished until it
completely vanishes (Fig. 3D).
This demonstrates that alternating activity in the entire swim CPG, as
evidenced by cyclic synaptic drive, is brought to a halt by hyperpolarizing a
single sint1. All of these observations were consistently made in
each of 32 separate whole animal preparations. Our interpretation of these
results is that sint1 is directly involved in pattern generation.
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Fig. 3. Graded interruption of swimming by increasing hyperpolarization of
sint1. (A–D) The upper traces in A–D show activity in
Rsint1 during a continuous episode of swimming. The lower traces show
the animal's swimming movements. Hyperpolarizing currents were applied
via the recording electrode using a bridge circuit. The timing of
current pulses is indicated by solid bars drawn under the behavioral record,
and the strength of the hyperpolarizing current is indicated under each bar.
Changes in absolute membrane voltage during stimulation are inaccurate because
of errors in bridge balance.
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Stimulation of sint1 shifts the phase of swimming
One of the strongest criteria one can use to determine whether a particular
neuron is a member of the CPG for a rhythmic behavior is a demonstration that
the phase of the behavior can be shifted in a predictable manner by altering
the firing pattern in the neuron (Friesen,
1994). We designed an experiment to test this as follows. A single
microelectrode was used to record from sint1 and to apply a constant
current pulse of sufficient amplitude to control its activity in a swimming
animal. The time of onset of the current pulse relative to the swimming cycle
and the duration of the pulse were varied. In the experiments illustrated in
Fig. 4, Lsint1 was
current-clamped while cellular activity and swimming behavior were recorded.
The period of the normal swimming cycle, defined as the interval between the
midpoints of adjacent spike bursts in Lsint1, was measured during ten
cycles of behavior immediately preceding the stimulus and the means ±
S.D. of the period calculated. These values were used to predict
the expected time of occurrence of the Lsint1 burst projected forward
in time beyond the period of stimulation. This allowed us to compare the
actual time of occurrence of succeeding bursts after the stimulus with the
expected time of occurrence calculated from the activity pattern prior to the
stimulus. Using this comparison, we could determine whether stimulation of
Lsint1 caused the output of the CPG to experience a phase shaft.
Predictions from the experiment are quantitative because we can ask whether
the phase shift is accurately predicted simply from knowledge of the stimulus
duration and its time of onset. In addition, we can ask whether phase shifts
produced by stimulating sint1 are permanent or whether the behavior
relaxes back into the original phase relationship over time. A permanent phase
shift is only expected if the stimulus resets the swim CPG.
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Fig. 4. Resetting the phase of swimming by stimulating sint1. The firing
pattern in Lsint1 was recorded along with a record of behavior (upper
trace in each panel) during a continuous episode of swimming. (A–C)
Hyperpolarizing current pulses sufficient to prevent soma and axon spikes in
Lsint1 were applied via the recording electrode. The timing
and duration of pulses were varied (pulse timing indicated by solid lines
below the voltage traces). In each experiment, the interval between the
centers of Lsint1 bursts was measured during the 10 cycles preceding
the onset of the stimulus (indicated by vertical strokes above the voltage
recording). These measurements were used to calculate the means ±
S.D. of the swim period, which were then used to predict the time
of occurrence of the Lsint1 burst projected forward in time beyond
the period of stimulation, based on the assumption that stimulation of
Lint1 has no effect on pattern generation. The predicted times of the
center of Lsint1 bursts are shown as vertical lines above the voltage
recording, with the S.D. represented by horizontal tics through the
lines. The results show that the assumption fails and that the phase of
swimming behavior is reset by the stimulus. The difference between the
predicted and actual time of occurrence of the burst provides a quantitative
measure of phase resetting (see details in the text). (D) The experiment was
repeated with an extra burst driven in Lsint1. The extra burst also
reset the phase of swimming in a quantitative fashion (see text). The bridge
circuit used to deliver stimulating currents was imperfectly balanced and,
therefore, the absolute membrane voltage is not accurately represented during
periods of stimulation. This experiment was conducted 28 times in four
different whole animal preparations with consistent results.
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In Fig. 4A, Lsint1
was hyperpolarized for a time equal to the swimming period (T) by a
current pulse beginning 0.47T after the peak contralateral bending
movement. The current was sufficient to prevent action potentials in both the
soma and axon. The result was that the behavior, measured two cycles after the
stimulus, was delayed by a factor of 0.46T, the delay expected
(0.47T) if the swim CPG had stopped for the duration of the
hyperpolarization and then re-started when the hyperpolarization ended. In
Fig. 4B, the cell was
hyperpolarized for 1.25T by a current pulse that began 0.38T
after the peak of the contralateral movement. The hyperpolarization delayed
the swimming rhythm by a factor of 0.62T, the delay expected
(0.63T) if the pattern generator was halted by the hyperpolarization.
In the third example (Fig. 4C), the cell was hyperpolarized for 3.2T beginning 0.38T after
the peak bending movement. This caused a phase shift of 0.6T, again
close to the expected value of 0.58T. Similar phase shifts occur when
sint1 is depolarized to drive an out-of-phase burst
(Fig. 4D). Because the phase
shift on stimulation of sint1 is predicted exactly from the stimulus
timing and duration, occurs in response to a single stimulus pulse, and
because the phase shift is maintained after the stimulus ends, we conclude
that sint1 is an integral member of the Melibe swim CPG.
Mutual inhibition between sint1 interneurons
The two sint1 neurons on opposite sides of the brain interact over
mutually inhibitory synaptic pathways. Fig.
5A,B shows that spike bursts driven in sint1 result in
hyperpolarization of the homologous cell on the opposite side of the brain.
Fig. 5C,D illustrates what
appears to be a unitary ipsp recorded in Lsint1 in response to either
a single action potential or a series of seven action potentials in
Rsint1. The apparent synaptic delay is 40 ms, which suggests either a
polysynaptic pathway or a monosynaptic connection that involves substantial
conduction time. We can gain some insight into the nature of this pathway from
LY dye-fills, which show that sint1 projects to the ipsilateral pedal
ganglion but does not project directly to the opposite pleural ganglion
via the dorsal commissure. It appears, therefore, that if the
inhibitory connections between the two sint1 values are monosynaptic,
the synaptic contact would have to be made in the pedal ganglion neuropil and
the axons of sint1 interneurons would have to course through the
ipsilateral pedal ganglion and continue to the opposite side via the
circumesophageal connective. This long path would involve considerable
conduction time and might explain a synaptic delay of 40 ms. The evidence from
LY dye-fills is inconclusive on this point. We did not see a process of
sint1 that projects all the way to the opposite pedal ganglion,
although this would be a difficult result to achieve since it is unlikely that
the dye could travel over the entire distance in a thin axonal process.
Another possibility is that the inhibitory connection is polysynaptic and
involves other interneurons. We describe a second class of interneurons,
termed sint2, which could in principle fill this role but caution
that there may be still others that have not yet been identified.
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Fig. 5. Reciprocal inhibition between sint1 neurons. (A,B). Simultaneous
recordings from Rsint1 (upper trace) and Lsint1 (lower
trace) in a quiescent whole animal preparation. In A, Rsint1 was
driven to fire a burst of action potentials while the voltage in
Lsint1 was recorded at 4 x higher gain. The same procedure was
followed in B, where Lsint1 was stimulated. (C,D). Unitary ipsp in
Lsint1 during stimulation of Rsint1. Rsint1 was driven to
fire either a single action potential (C) or a series of seven action
potentials at a rate of one per second (D, sweeps superimposed). Asterisks
indicate the time of occurrence of the ipsp. This experiment was repeated in
six different whole animal preparations with consistent results.
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Output from sint1 to motoneurons
Sint1 makes excitatory synaptic connections with the motoneurons
in the ipsilateral pedal ganglion that are responsible for contraction of the
ipsilateral body musculature during swimming.
Fig. 6 shows epsps in eight
different right pedal ganglion motoneurons in response to action potentials
driven in Rsint1. The epsps in
Fig. 6C–H appear to be
monosynaptic because they follow sint1 action potentials at
frequencies in excess of 10 Hz and occur with latencies of 2–5 ms
(measured from the peak of the presynaptic action potential recorded in the
soma to the foot of the epsp). The epsps in
Fig. 6A,B occur with longer
latencies (20 and 40 ms, respectively) and may represent excitatory input to
motoneurons over polysynaptic pathways. Even in these examples, however,
synaptic transmission did not fail during repetitive stimulation at
frequencies like those seen during swimming. All of the synaptic potentials
illustrated in Fig. 6 appear to
be chemically mediated because in each case the amplitude of the epsp
increased when the postsynaptic cell was hyperpolarized. There is considerable
variability in the rise time, amplitude, and duration of epsps recorded in
different motoneurons. It is not known whether this involves presynaptic or
postsynaptic mechanisms but it is apparent that action potentials in
sint1 give rise to postsynaptic potentials in follower cells that
differ in amplitude and time course. These differences undoubtedly contribute
to the characteristic differences in the firing patterns of individual
motoneurons during swimming.
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Fig. 6. Excitatory synaptic output from sint1 to synergistic motoneurons.
The dorsal aspect of the right half of the CNS, including the right pleural
and pedal ganglia, is shown diagrammatically (rostral direction upward). The
diagram also shows the relative positions of the soma of Rsint1 in
the pleural ganglion and of eight individual motoneurons in the ipsilateral
pedal ganglion that are known to participate in swimming behavior. Single
spikes were driven in Rsint1 at a rate of one per second in a
quiescent whole animal preparation while recording membrane voltage at higher
gain in the motoneurons. The results from 5–10 repetitions are
superimposed. In each case the spike in Rsint1 elicits an epsp in the
motoneuron. The time calibration bar (near A) corresponds to 40 ms
(A–C,G) and 20 ms (D–F,H). For the postsynaptic potentials, the
vertical calibration corresponds to 2 mV (A,B), 4 mV (F–H), and 20 mV in
the other recordings. The epsps in A and B occur with latencies of 20–40
ms, while those in C–H occur with latencies of 2–5 ms. These
results are characteristic of those obtained in 30 separate whole animal
preparations involving >150 paired recordings.
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Sint1 inhibits antagonistic swim motoneurons in the opposite pedal
ganglion. Fig. 7A shows a
simultaneous recording from Rsint1 and a motoneuron in the left pedal
ganglion in a quiescent whole animal preparation. When Rsint1 was
driven to fire a burst, the motoneuron was strongly hyperpolarized. Similar
results were obtained in isolated ganglion preparations (N=5). We
found no evidence for direct or indirect synaptic feedback from synergistic or
antagonistic motoneurons to sint1 in any of 32 experiments employing
simultaneous intracellular recording. Single action potentials or sustained
bursts driven in synergistic or antagonistic motoneurons did not produce
discernible psps or change the voltage recorded in sint1 (example in
Fig. 7B). When sint1
was depolarized with constant current sufficient to drive low frequency
repetitive firing in a quiescent animal, the firing frequency was not altered
by driving strong bursts in motoneurons. The conclusion we draw is that while
sint1 is presynaptic to many of the motoneurons involved in swimming,
its activity is not influenced by synaptic feedback from motoneurons.
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Fig. 7. Inhibitory synaptic output from sint1 to an antagonistic
motoneuron recorded in a quiescent whole animal preparation. (A) A driven
burst of action potentials in Rsint1 (lower trace) causes
hyperpolarization of an identified antagonistic motoneuron located in the
opposite pedal ganglion. (B) A driven burst in the motoneuron has no effect on
Rsint1.
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Swim interneuron 2 (sint2)
A second type of interneuron, termed swim interneuron 2 (sint2),
shares many of the properties of sint1. Two cells of this type have
been identified, one on the dorsal surface of each pedal ganglion (see
Fig. 1). The pattern of
activity in Lsint2 during a brief episode of swimming is shown in
Fig. 8A. When swimming was
initiated by withdrawing a surface from the foot at the first arrow,
Lsint2 began to fire in bursts that begin before and continue
throughout most of the bending movement to the ipsilateral side. When swimming
was terminated by returning the surface at the second arrow, bursting activity
ceased and sint2 resumed irregular firing. The action potential burst
in sint2 occupies 49.7±8.1% (N=9) of the swim period.
Using the time of maximum ipsilateral bending as a reference, the action
potential burst in sint2 begins 206.8±6.6° (N=5)
before the peak of the ipsilateral movement and ends 4.5±17.4°
(N=5) before the peak, a phase relationship that is maintained during
long episodes of swimming. From these measurements it is clear that the
beginning of the burst in sint2 lags the beginning of the burst in
the synergistic sint1 by about 43° and that sint2
continues to fire for a greater fraction of the period (see
Fig. 15). The latency between
the beginning of the burst in sint1 and the beginning of the burst in
the synergistic sint2 was measured in dual microelectrode experiments
and found to be 460±124 ms (N=3).
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Fig. 8. Firing pattern in swim interneuron 2 (sint2). (A) Activity in
Lsint2 during a brief episode of behavior. Swimming was initiated by
removing a surface from the animal's foot at the first arrow and terminated by
replacing the surface at the second arrow. (B) Action potential bursts in
Lsint2 shown at higher gain and on an expanded time scale. (C)
Alternating bursts in the two antagonistic sint2 neurons recorded
simultaneously during a long episode of swimming.
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Fig. 15. The timing of bursts in sint1 and sint2 during swimming.
(A,B) Simultaneous recording from Rsint1 (A) and Rsint2 (B)
during an episode of swimming in a whole animal preparation. After this
recording was taken, the electrode was removed from Rsint1 and
Lsint1 was impaled. (C,D) Simultaneous recording from Rsint2
(C) and Lsint1 (D) during the same swimming episode. The two sets of
records were aligned to the midpoint of the center burst in Rsint2 so
that the timing of bursts could be compared. All recordings are from the same
whole animal preparation. Similar results were obtained in three separate
whole animal experiments.
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The structure of action potential bursts in sint2 is shown on an
expanded scale in Fig. 8B.
During swimming, bursts appear to be driven by strong excitatory synaptic
input while the intervals between bursts are characterized by prominent ipsps.
A simultaneous recording from both sint2 neurons during an episode of
sustained swimming is shown in Fig.
8C. The two cells fire in antiphase and the end of the burst in
one sint2 either does or does not overlap the beginning of the burst
in the contralateral homologue. The bursts in sint2 have a
complicated substructure, but these recordings suggest that the two cells may
share synaptic inputs from some of the same sources. For example, when there
is a pause in the burst in one sint2, its homologue experiences an
abrupt depolarization. This suggests that there may be presynaptic neurons
that simultaneously excite one sint2 and inhibit the other. We
demonstrate below that sint1 has precisely these properties.
Effect of sint2 on swimming behavior
Sint2 resembles sint1 in its influence on swimming
behavior. When a single sint2 is driven to fire a burst while the
animal is crawling on a surface, the animal bends toward the ipsilateral side
for the duration of the stimulus. The driven movement resembles the normal
ipsilateral swim movement. Similarly, when sint2 is driven to fire a
sustained burst while the animal is swimming, the behavior is interrupted and
the animal remains contracted to the stimulated side. Hyperpolarizing
sint2 to prevent firing during swimming also interrupts the behavior,
causing the animal to remain contracted to the opposite side.
Stimulation of sint2 also resets the phase of swimming. We
performed an experiment identical to the one used to show phase resetting on
stimulation of sint1. In the example in
Fig. 9, Lsint2 was
hyperpolarized with current sufficient to block soma and axon spikes for 3.8 s
(equivalent to 0.51 times the period in the freely swimming animal) while
simultaneously recording intracellular activity and swimming behavior. The
current pulse began 622 ms after the end of a sint2 burst and caused
the animal to spend more time contracted to the unstimulated side, lengthening
the interburst interval by 2.1 s. The result was a delay in the swimming
rhythm (measured two cycles after the stimulus) of 2.3 s, very close to the
delay expected if the CPG for swimming had been halted as long as
sint2 was hyperpolarized and then resumed its activity immediately
after the hyperpolarization ended. Phase resetting was also observed when
sint2 was driven to produce a novel burst of action potentials (not
shown). Similar results were obtained in 3 separate whole animal preparations.
Our interpretation of these results is that sint2 is also an integral
member of the CPG for swimming.
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Fig. 9. Resetting the phase of swimming by stimulating sint2. (Top) An
intracellular recording from Lsint2 during swimming; (bottom) a
record of the animal's movements. The movement detector saturated and did not
report the full range of side-to-side movement in this example.
Hyperpolarizing current sufficient to silence the cell was applied
via the recording electrode during the time indicated by the bar
under the voltage recording. The time of maximal right flexion (shown by
vertical lines above the behavior record) was measured during the 10 cycles
preceding the onset of the stimulus in order to calculate the mean ±
S.D. of the swimming period. These values were projected forward in
time to predict the expected time of occurrence of maximal right flexion The
bars and vertical lines above the behavioral record show the predicted time of
occurrence of peak right flexion after the stimulus ends. Horizontal tics show
the S.D. The difference between the predicted time of occurrence
and the actual occurrence of peak flexion provides a measure of phase
resetting. This experiment was repeated 15 times in three different whole
animal preparations with consistent results.
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Electrical coupling between synergistic interneurons sint2 and sint1
Sint2 is electrically coupled to the synergistic sint1 by
a non-rectifying electrical synapse. Fig.
10 illustrates simultaneous microelectrode recordings from
Rsint1 and Rsint2. It shows that d.c. current is conducted
symmetrically in both directions and that the junction is characterized by a
d.c. coupling coefficient of 0.1 at voltages near the resting potential
(Fig. 10A). Action potentials
are not coupled symmetrically, however, and conduction in the direction
sint1 to sint2 is much stronger
(Fig. 10B). This asymmetry can
be expressed in terms of a spike-coupling coefficient, defined as the maximum
amplitude of the electrical psp divided by the amplitude of the presynaptic
action potential. With this definition, the spike-coupling coefficient for
conduction from sint1 to sint2 is 0.03 while the coupling
coefficient in the opposite direction, from sint2 to sint1
is nearly ten times less (0.005).
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Fig. 10. Electrical coupling between sint1 and the synergistic
sint2. (A,B) Simultaneous recordings from Rsint1 (upper
traces) and Rsint2 (lower traces) in a quiescent whole animal
preparation. Hyperpolarizing current pulses were applied to one of the cells
via the recording electrode while membrane voltage was recorded in
the other. Bridge balance was checked using short hyperpolarizing pulses of
the same amplitude before and after the recording. (Ai) A current pulse
applied to Rsint1 causes hyperpolarization of Rsint2 due to
current flow across the electrical junction. (Aii) The reciprocal connection.
(Bi) The electrically coupled epsp in Rsint2 (lower trace; recorded
at higher gain) in response to driven action potentials in Rsint1
(eight superimposed traces, stimulus rate one per second). (Bii) The strongly
attenuated electrically coupled epsp recorded in Rsint1 (upper trace;
recorded at higher gain) in response to single action potentials driven in
Rsint2 (seven superimposed traces, stimulus rate one per second). (C)
Bode plot showing conduction of sinusoidal currents across the electrical
junction in both directions. Rsint1 and Rsint2 were
stimulated, one at a time, with subthreshold currents of fixed amplitude but
varying frequencies while recording the coupled sine wave in the other cell.
The electrical junction passes current symmetrically in both directions and
has the characteristics of a low-pass filter with a corner frequency of 1.5 Hz
and final slope of 6 dB per octave in frequency. These results were confirmed
in each of six separate whole animal preparations.
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Asymmetrical spike coupling could result from several causes. Because the
d.c. coupling coefficient is the same in both directions, it would appear that
the asymmetry reflects a capacitative term, possibly due to the physical
location of the junction relative to the stimulating and recording sites. To
test this idea, transfer functions characterizing the electrical junction were
measured in both directions. One cell was driven with constant amplitude,
subthreshold, sinusoidal current of varying frequency via a somatic
microelectrode, while the electrically coupled sine wave was recorded in the
soma of the other cell. The experiment was then repeated after switching the
current source. The normalized amplitudes of coupled sine waves for
transmission in both directions are plotted in
Fig. 10C. The transfer
functions are identical and show that the junction has the characteristics of
a low-pass filter with a cut-off frequency of 1.5 Hz and final slope of 6 dB
per octave in frequency. From these measurements we can conclude that
asymmetrical spike coupling does not result from differences in cell input
capacitance or junctional capacitance. The most likely explanation is that the
asymmetry has an anatomical basis. Results from LY dye-fills show that
sint1 sends an axonal projection to the ipsilateral pedal ganglion
but sint2 does not project to the pleural ganglion, indicating that
the electrical junction must be formed in the pedal ganglion neuropil. With
this arrangement a spike originating near the cell body of sint1 is
expected to propagate actively over much of the pathway and produce a relative
large electrical psp in the soma of sint2. A spike originating near
the cell body of sint2, however, would propagate actively over only a
fraction of the distance, spreading passively the rest of the way, and is
expected to produce a more attenuated psp in the soma of sint1. An
important consequence is that the electrically coupled psp is expected to have
a significant effect on the excitability of sint2 because it occurs
close to the site of spike initiation. In contrast, an electrical junction
located far from the spike initiation zone of sint1 would be expected
to have little effect on its excitability. This anatomical arrangement may
allow sint1 and sint2 to function independently under
conditions in when sint2 receives excitatory synaptic input that is
not shared with the synergistic sint1.
Mutual inhibition between sint2 neurons
The two sint2 neurons form mutually inhibitory synaptic
connections. When either cell is driven to fire a burst of action potentials
(Fig. 11), its homologue in
the opposite pedal ganglion receives strong inhibitory input that begins after
a delay (42.8±15.1 ms; N=5). The summed inhibitory potential
has a prolonged time course, decaying with a half time of 342±23.9 ms
(N=5) after the stimulus ends. This slow decay could be explained
either by the presence of interposed interneurons or by prolonged transmitter
action. In addition, the inhibitory pathway is somewhat labile. When
sint2 is driven to fire a burst of action potentials at a frequency
like that seen during a swimming burst (e.g. 10–16 Hz) theamplitude of
the summed ipsp in the contralateral sint2 declines to 70% of its
peak amplitude with a half-time of 4.5 s. This decline suggests a synaptic
fatigue process that may play a role in timing oscillation in the network.
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Fig. 11. Mutual inhibition between antagonistic sint2 neurons. Simultaneous
recordings from Lsint2 and Rsint2 in a quiescent whole
animal preparation. (A) Lsint2 (upper trace) was driven to fire two
bursts of action potentials while recording membrane voltage in
Rsint2 (lower trace) at higher gain (time of stimulus marked by bar).
(B) Stimulation of Rsint2 to fire three bursts while recording from
Lsint2. Action potentials were truncated by the recording device in
the high gain records. The firing frequency during driven bursts was similar
to what is seen during normal swimming (see
Fig. 8). Similar results were
obtained in each of five whole animal experiments.
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Mutual inhibition between sint2 and the contralateral sint1
Sint2 also forms mutually inhibitory synaptic connections with the
antagonistic sint1 located in the pleural ganglion on the opposite
side of the brain. Fig. 12A
shows the ipsp recorded in Rsint1 when Lsint2 was driven to
fire single action potentials. The synaptic connection in the opposite
direction is illustrated in Fig.
12B. The ipsps are similar in amplitude and duration at a given
postsynaptic voltage and the inhibitory pathways do not fail at stimulus rates
as high as 10 Hz. When the presynaptic cell is stimulated to fire a burst that
matches the frequency and duration of the swimming burst, the summed ipsp does
not decline, indicating that the pathway is resistant to fatigue. Ipsps follow
presynaptic spikes with latencies of 35 to 45 ms, suggesting either a
polysynaptic pathway or significant conduction time. LY dye-fills show that
sint1 sends a process to the ipsilateral pedal ganglion where it
arborizes. The antagonistic Sint2 projects to the same pedal ganglion
neuropil via the pedal-pedal connective. This means that if the
mutually inhibitory connections between sint2 and the contralateral
sint1 are monosynaptic, they are likely to be made in the neuropil of
the pedal ganglion on the same side as the sint1 cell body. In this
case, conduction through the sub-esophageal connective might explain the long
synaptic delay.
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Fig. 12. Mutual inhibition between the antagonistic sint1 and
sint2. Simultaneous recordings from Lsint2 (upper traces)
and Rsint1 (lower traces) in a quiescent whole animal preparation.
(A) Single action potentials were driven in Lsint2 at a rate of one
per second while recording from Rsint1 at higher gain (7 superimposed
traces). (B) The reciprocal ipsp in Lsint2 during stimulation of
single action potentials in Rsint1 at 1 Hz (6 superimposed traces).
Similar results were obtained in each of 12 separate whole animal
experiments.
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Output from sint2 to motoneurons
Sint2 makes excitatory synaptic connections with synergistic
motoneurons and inhibitory connections with antagonists.
Fig. 13 shows results of
experiments using an isolated brain preparation. In
Fig. 13A, sint2
(lower trace) was driven to fire a burst of action potentials while recording
from a synergistic motoneuron in the same pedal ganglion. There is one-for-one
correspondence between spikes in sint2 and epsps in the motoneuron.
Fig. 13B shows the
relationship between activity in sint2 (lower trace) and an
antagonistic motoneuron located in the opposite pedal ganglion during fictive
swimming. Again, there is close correspondence between action potentials in
the interneuron and individual ipsps in the motoneuron. Similar results were
obtained in whole animal preparations.
Fig. 14A illustrates what
appear to be unitary ipsps in a pedal ganglion motoneuron in response to
driven spikes in the contralateral sint2. The postsynaptic potential
begins with a delay of 35–45 ms after the peak of the spike in the
interneuron. A significant fraction of the delay must be the result of
conduction time since the only known pathway between sint2 and the
neuropil of the opposite pedal ganglion is via the subesophegeal
pedal-pedal connective. Fig.
14B illustrates summation of ipsps in the motoneuron when
sint2 was driven to fire a burst of action potentials. The inhibitory
connection is not reciprocal because a driven burst in the motoneuron had no
effect on sint2 (not shown).
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Fig. 13. Synaptic output from sint2 to motoneurons. (A) Excitatory output
from sint2 to a synergistic motoneuron recorded in a quiescent
isolated brain preparation. Sint2 (lower trace) was driven to fire a
burst of action potentials by direct stimulation while recording membrane
voltage in the motoneuron (upper trace). Individual spikes in sint2
correspond with unitary epsps in the motoneuron. (B) Ipsps in an antagonistic
motoneuron coincident with action potentials in sint2 during fictive
swimming in an isolated brain preparation.
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Fig. 14. Inhibitory output from sint2 to an antagonistic motoneuron. (A)
Single action potentials were driven in sint2 at a rate of one per
second while recording from an antagonistic motoneuron in the opposite pedal
ganglion in a quiescent whole animal preparation. Two traces are superimposed.
The ipsp occurs with a delay of about 35 ms. (B) A driven burst in
sint2 causes sustained hyperpolarization in the motoneuron due to
summation of ipsps. These results are characteristic of 7 separate
experiments.
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Timing of bursts in the interneurons during swimming
The temporal relationships between activity in the interneurons during
swimming are illustrated in Fig.
15. The figure shows pair-wise recordings from Rsint1 and
Rsint2 (two upper traces) and from Rsint2 and
Lsint1 (two lower traces). The recordings were obtained from the same
whole animal preparation during a single experiment and are aligned on the
action potential bursts in Rsint2 (see
Fig. 15 legend). There are
three important timing relationships to note. (1) The burst in sint1
begins before the burst in the synergistic sint2 and ends before the
sint2 burst ends. (2) In this example, activity in sint1
begins somewhat after the end of the burst in the antagonistic sint2
and there is no overlap. This is not always the case, however, and the burst
in sint1 can begin during the last one or two spikes in the
contralateral sint2 burst. (3) Bursts in the two antagonistic
sint1 neurons alternate without overlap. This last point has an
important consequence. It was inferred from
Fig. 2 that burst termination
in sint1 coincides with an increase in ipsp input yet the
antagonistic sint1 and sint2, the two cells known to inhibit
sint1, do not fire at this time. This raises the possibility that
there may be additional neurons participating in swim generation that have not
yet been identified.
Activity in Sint1 and Sint2 dissociates during other locomotor behaviors
Sint1 is normally silent while the animal is crawling on a surface
and it is largely insensitive to sensory input, firing at most a few spikes in
response to tactile stimulation of the body. In contrast, sint2 fires
sporadically in crawling animals, exhibiting periods of sustained firing along
with periods of silence. Sustained firing is always correlated with turning
movements toward the ipsilateral side and sint2 receives prolonged
inhibitory input during turning toward the opposite side.
Fig. 16A shows simultaneous
recordings from Rsint1 and Rsint2 during turning toward the
right in response to a tactile stimulus applied to the left body wall of an
animal that was crawling on a seaweed surface. Rsint2 (lower trace)
fires throughout the turning movement and it is apparent that this is the
result of a sustained increase in epsp input. The synergistic sint1
(upper trace) also receives excitatory synaptic input, especially at the
beginning of the movement, but the input is subthreshold and sint1
does not fire. It would appear that even though sint1 is inhibited by
foot contact, it nevertheless receives subthreshold excitation during turning
movements toward the ipsilateral side. Activity in the same two cells during a
spontaneous turning movement toward the right is shown in
Fig. 16B. It appears that
sint2 participates in both spontaneous and stimulated turning while
sint1 fires weakly or not at all during turning. This suggests that
the CPG for swimming is formed dynamically, when activity in the
sint1 and sint2 cell pairs becomes bound together. When this
does not occur, the same interneurons appear to function independently during
the performance of other behaviors that involve the same or similar
musculature, such as turning.
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Fig. 16. Recordings from sint1 and sint2 during turning movements.
(A) A turning movement to the right was initiated by a tactile stimulus
applied to the left side of the body while recording from Rsint1
(upper trace) and Rsint2 (lower trace). (B) Activity in the same two
cells during a spontaneous turn to the right. A and B are from a whole animal
preparation that was allowed to crawl on a blade of seagrass during the
recording. Similar observations were made in each of 12 separate whole animal
experiments.
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Discussion
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The synaptic interactions between the four interneurons we identified as
members of the Melibe swim CPG are summarized diagrammatically in
Fig. 17. Interneurons on the
same side of the midline are electrically coupled, while those on opposite
sides are linked by mutually inhibitory connections. The major criterion we
used to identify members of the swim CPG was the ability to permanently shift
the phase of swimming by stimulating individual neurons. Both sint1
and sint2 meet this criterion and they share a number of other
features that suggest that they work together to generate the behavior. Both
types of interneurons fire in bursts that are phase-locked with swimming and
precede the ipsilateral swimming movement. Both have a strong and lasting
effect on behavior since a brief depolarizing or hyperpolarizing current
applied to any of them is sufficient to cause a permanent phase shift. In
addition, stimulation of any of the interneurons in a resting animal causes
the animal to bend to the ipsilateral side, producing a coordinated movement
that resembles the swimming movement toward that side. When sint1 or
sint2 is driven to fire a long burst in a swimming animal, the
behavior is interrupted and the animal remains contracted to the stimulated
side; conversely, when the cell is hyperpolarized to prevent firing during
swimming the animal remains contracted to the opposite side. Finally, the
output from both sint1 and sint2 is distributed in a manner
appropriate for members of the swim CPG, exciting synergistic motoneurons and
inhibiting antagonists, thus providing the reciprocal drive onto motoneurons
necessary to generate alternating side-to-side movements. Following the
reasoning of Friesen (1994) and
Svoboda and Fetcho (1996) we
take these findings as strong evidence that the two sint1 and the two
sint2 neurons are integral members of the CPG for Melibe
swimming. The experiment in which sint1 was progressively
hyperpolarized during swimming (Fig.
3) led to an additional important observation. It appears that
strong hyperpolarization of a single sint1 abolishes the periodic
synaptic drive that interneurons within the CPG normally receive during
swimming. Apparently, hyperpolarization of a single member of the network can
stop rhythmic activity in the entire network (see also Figs
4,
9). It appears, therefore, that
the CPG for swimming functions when activity in all four interneurons is bound
together and the electrical connections between synergistic interneurons may
help to establish this grouping.
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Fig. 17. Network model for the Melibe swim CPG. The mutually inhibitory
connections linking the left (L) and right (R) sint1 and
sint2 neurons are shown by lines terminating in circles. The
electrical synapses between the synergistic sint1 and sint2
neurons are shown by lines terminating in bars.
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Although sint1 and sint2 fire in a coordinated fashion
during swimming, they have very different firing patterns and serve different
roles in the generation of other behaviors. When the animal is crawling on a
surface sint1 is silent, perhaps because it receives inhibitory input
from sensory pathways signaling foot contact, but sint2 is not silent
and fires irregularly. Furthermore, sint2 begins to fire during the
initiation of turning movements toward the ipsilateral side and continues to
fire throughout turning while the synergistic sint1 remains silent.
It would appear that sint2 is multifunctional, participating in at
least two locomotor behaviors (swimming and turning), while sint1
participates only in swimming. Although the synergistic sint1 and
sint2 are electrically coupled, they can work independently under
certain conditions because the anatomical arrangement favors conduction of
action potentials in only one direction, from sint1 to
sint2. This feature may allow the network to rearrange in a dynamic
fashion, dictated by the nature of the synaptic input from other sources. In
this way, part of the motor system responsible for generating side-to-side
swimming movements can be used to orchestrate non-rhythmic, unilateral
movements that involve the same musculature. Even during turning, however, the
mutually inhibitory connections between sint2 neurons ensure that
when synergistic motoneurons are excited, antagonists will be inhibited. This
organization is reminiscent of the situation in Tritonia where the
CPG for escape swimming can act in different states of coordination to serve
different functions (Katz et al.,
1994,
2001).
We observed that when the foot loses contact with a surface, sint1
immediately depolarizes to a level 5–10 mV above the voltage recorded in
the quiescent animal. Spike bursts in sint1 are superimposed on this
depolarization (Fig. 2A). This
feature is unique to sint1 and is not seen in sint2 or in
the motoneurons. The sustained depolarization in sint1 may provide an
important clue into how the CPG is dynamically structured and it is possible
that control over the initiation of swimming involves only removal of
inhibition at the level of sint1, a situation reminiscent of the role
of tarsal inhibition in locust flight
(Ritzmann et al., 1980). This
has not yet been demonstrated by direct experiment, but it raises the
interesting possibility that a change in sensory input might cause dynamic
restructuring of the CPG network.
The swimming motor system appears to be hierarchically arranged. While
sint1 and sint2 both fire bursts during swimming, the timing
of the bursts in the two classes of interneurons is characteristically
different. The sint1 burst always begins before the burst in the
synergistic sint2 and ends before the end of the sint2
burst, a sequence that persists even though sint1 and sint2
are electrically coupled. Apparently the electrical synapse is not strong
enough to fully synchronize their activity. Spike bursts in the two
sint1 neurons do not overlap while bursts in the two antagonistic
sint2 neurons may or may not overlap. Finally, termination of
swimming is correlated with cessation of firing in sint1 but not with
silencing of sint2. These observations suggest that the two types of
interneurons occupy different positions in a hierarchically arranged motor
system. Because sint1 appears to be active only during swimming, we
think of it as a key element in the swim CPG and that without its
participation, the network cannot function in the alternating mode
characteristic of swimming. At still another level, the motoneurons appear to
act as followers whose activity is determined by input from interneurons
(Watson et al., 2002). There
are between 14 and 21 motoneurons in each pedal ganglion that participate in
swimming (S. H. Thompson, unpublished observations) but there is no evidence
that any of the motoneurons feed back to interneurons, a finding consistent
with the idea that motoneurons function as a final common path for locomotor
behavior but do not participate in pattern generation.
Where are the inhibitory connections between interneurons made?
Swim interneurons on opposite sides of the brain make reciprocal inhibitory
connections, an organization that provides a plausible mechanism for the
production of alternating activity in the network
(Perkel and Mulloney, 1974;
Marder and Eisen, 1984;
Getting, 1989a; Satterlie,
1985,
1989;
Pearson, 1993;
Friesen, 1994). There is
uncertainty, however, about where the inhibitory connections are made and
whether they are monosynaptic or polysynaptic. Resolving these issues will be
important in order to fully understand which features of network architecture
are responsible for determining its output frequency and stability.
Sint2 projects to the opposite pedal ganglion via a
process that runs in the sub-esophageal pedal-pedal connective. We
consistently found that alternating activity in the swim CPG, and swimming
behavior, ceases when this pathway is cut (N=5; W. Watson,
unpublished observations). We interpret this as strong evidence that axons
traveling in the sub-esophageal connectives are necessary for the expression
of oscillating activity in the network, the most likely reason being that
these axons are responsible for the mutually inhibitory interactions between
interneurons. The apparent synaptic delay for the inhibitory interactions
(35–45 ms) could be the result of conduction time or it could indicate
that the pathways are polysynaptic. Some insight into conduction time over the
sub-esophageal connectives can be gained from the following observation. A
synaptic delay of 40–45 ms was measured for an apparently monosynaptic
inhibitory connection between sint2 and an antagonistic motoneuron in
the opposite pedal ganglion. Evidence from dye-fills indicates that this
synapse is most likely to be made in the neuropil that contains the motoneuron
cell body by an axonal process of sint2 traveling to that neuropil
via the pedal-pedal connective. If the axons responsible for mutual
inhibition between int2 values follow the same route, then a synaptic
delay of 40–45 ms is not inconsistent with a monosynaptic
connection.
The pathway responsible for mutual inhibition between the two
sint1 interneurons is less clear. In anatomical studies,
sint1 was never found to project to the opposite pleural ganglion
via the central commissure. It is possible that sint1
projects to the contralateral side through the sub-esophageal connective,
although this too was not seen in dye-fills. An alternative suggestion is that
the mutual inhibition between sint1 neurons involves a polysynaptic
pathway, and there are two possibilities. Sint1 might first drive the
ipsilateral sint2, which in turn makes inhibitory synapses with the
antagonistic sint1 and sint2 in the neuropil of the opposite
pedal ganglion. The second possibility is that another as-yet-unidentified
interneuron mediates mutual inhibition between the two sint1
neurons.
Are sint1 and sint2 the only members of the swim CPG, or are there missing elements?
We identified four interneurons as integral members of the CPG for
Melibe swimming. The question remains, however, whether there are
additional neurons that we have not yet identified that contribute in
important ways to network function. Microelectrode sampling bias can never be
completely eliminated. Although we tried to minimize the problem by sampling
widely in the Melibe central nervous system, there may be other
important units that are missing from our analysis because they were not
observed or because their importance was not recognized. One observation in
particular suggests that additional neurons may be involved in pattern
generation. We are not able to explain the appearance of ipsps in
sint1 at burst termination (see
Fig. 2B) because none of the
interneurons demonstrated to have inhibitory interactions with sint1
are active at this time. Recent studies suggest that another interneuron,
anatomically distinct from sint1 and sint2, may play a role
in swim generation (J. Newcomb, personal communication). It is not known
whether this newly described neuron inhibits sint1 or sint2,
but determining if it has a role in the swim CPG and defining its function
will be important.
Comparison of the Melibe swim CPG with that of other opisthobranchs
Several opisthobranch species exhibit swimming behavior, some for escape
and some for locomotion. In five species there is specific knowledge of the
organization of the swim CPG (Clione limacine, Aplysia brasiliana,
Tritonia diomedea, Pleurobranchaea californica and now Melibe
leonina). Given the diversity of swimming modes and the wide phylogenetic
divergence between these five species, it is not surprising to find more
differences than similarities in swim CPGs
(Katz et al., 2001). The
Tritonia and Pleurobranchaea CPGs are the only two that have
clear similarities, possibly because the mode of swimming is so similar. Both
animals swim to escape from predators and their swimming activity is limited
to short bouts. Many homologous neurons have been identified in these two
species and the basic mechanisms underlying initiation, production and
termination of the swim rhythm are similar
(Getting, 1989a;
Jing and Gillette, 1999:
Gillette and Jing, 2001). The
interneurons identified in Melibe have features in common with the A4
and A10 interneurons in Pleurobranchaea, which also project to the
ipsilateral pedal ganglia and have major influence on the generation of
swimming (Jing and Gillette,
2003). Clione swims for locomotion rather than primarily
for escape. The swimming circuit in Clione resembles the swim CPG in
Melibe in that reciprocal inhibition appears to be the dominant
mechanism for pattern generation and key elements of the CPG appear to reside
in the pedal ganglia in both animals
(Satterlie, 1985;
Arshavsky et al., 1985).
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Acknowledgments
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We thank Dennis Willows, Peter Getting, Bradley Jones and Jim Newcomb for
helpful discussions and Jim Newcomb for assistance with anatomical studies.
Thanks also to Larry Cohen, Melissa Coates and Christian Reilly for comments
on the manuscript. We thank the staff of the Hopkins Marine Station and Friday
Harbor Laboratories for support and the numerous people who helped collect
Melibe at both locations. Supported in part by IBN-9514421 to S.T.
and NSOD36411 to W.W.
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References
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Arshavsky, Yu. I., Beloozerova, I. N., Orlovsky, G. N., Panchin,
Yu. V. and Pavlova, G. A. (1985). Control of locomotion in
marine mollusc Clione limacina. II. Rhythmic neurons of pedal
ganglia. Exp. Brain Res.
5
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Summary
Introduction
