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The Journal of Neuroscience, May 1, 1999, 19(9):3610-3619
Motor Pattern Specification by Dual Descending Pathways to a
Lobster Rhythm-Generating Network
Denis
Combes,
Pierre
Meyrand, and
John
Simmers
Laboratoire de Neurobiologie des Réseaux, Université
Bordeaux I and Centre National de la Recherche Scientifique,
Unité Mixte de Recherche 5816, 33405 Talence,
France
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ABSTRACT |
In the European lobster Homarus gammarus, rhythmic
masticatory movements of the three foregut gastric mill teeth are
generated by antagonistic sets of striated muscles that are driven by a neural network in the stomatogastric ganglion. In
vitro, this circuit can spontaneously generate a single (type
I) motor program, unlike in vivo in which gastric mill
patterns with different phase relationships are found. By using paired
intrasomatic recordings, all elements of the gastric mill network,
which consists mainly of motoneurons, have been identified and their
synaptic relationships established. The gastric mill circuit of
Homarus is similar to that of other decapod crustaceans,
although some differences in neuron number and synaptic connectivity
were found. Moreover, specific members of the lobster network receive
input from two identified interneurons, one excitatory and one
inhibitory, that project from each rostral commissural ganglion.
Integration of input from these projection elements is mediated by
synaptic interactions within the gastric mill network itself. In
arrhythmic preparations, direct phasic stimulation of the previously
identified commissural gastric (CG) interneuron evokes gastric mill
output similar to the type I pattern spontaneously expressed in
vitro and in vivo. The newly identified gastric
inhibitor interneuron makes inhibitory synapses onto a different subset
of gastric mill neurons and, when activated with the CG neuron, drives
gastric mill output similar to the type II pattern that is only
observed in the intact animal. Thus, two distinct phenotypes of gastric
mill network activity can be specified by the concerted actions of
parallel input pathways and synaptic connectivity within a target
central pattern generator.
Key words:
Crustacea; Homarus gammarus; stomatogastric
nervous system; central pattern generator; projection interneurons; network reconfiguration
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INTRODUCTION |
It is now well established that most
rhythmic motor behaviors are generated by neural networks, called
central pattern generators (CPGs), that can operate in the absence of
feedback from peripheral sense organs (Delcomyn, 1980 ). Nevertheless,
to be functionally meaningful in vivo, CPGs must be
susceptible to sensory and/or central modulatory input to adapt their
motor output to changing behavioral demands. Thus, a given motor
network must be able to alter its expression on a cycle by cycle basis,
as well as over prolonged periods of ongoing activity. Several
mechanisms by which a CPG can undergo functional reorganization have
been proposed (Getting and Dekin, 1985). Two of these rely on
modifications within the network itself, involving neuromodulation of
synaptic interactions among constituent neurons and/or alterations in
their intrinsic membrane properties (Marder and Calabrese, 1996).
A third mechanism involves changes in the way sensory inputs impinge on
the network, although a full understanding of this mechanism is
primarily lacking because of the complexity and diversity of the
afferent pathways involved (Soffe, 1997).
A preparation well suited to address this problem is the gastric mill
system controlling rhythmic masticatory movements in the foregut of
decapod crustaceans. Gastric mill movements are driven by the gastric
mill motor network of the stomatogastric nervous system (STNS). This
network can remain spontaneously active in vitro, and in
several species the cellular properties and synaptic connectivity of
its constituent neurons are known in detail (Selverston and Moulins,
1987; Harris-Warrick et al., 1992). Although a number of modulatory
input pathways and their long-lasting influences on the gastric mill
CPG have been elucidated (Meyrand et al., 1994; Norris et al., 1994;
Blitz and Nusbaum, 1997), the role of "conventional" synaptic
inputs, such as those arising from movement-related sensory feedback,
in mediating rapid short-term reorganization of gastric mill motor
output is still poorly understood.
In the present in vitro study, we describe the synaptic
wiring of the previously incompletely known gastric mill CPG of
the European lobster Homarus gammarus and analyze the
influence of two identified interneurons that project from more rostral
ganglia. One of these projection interneurons, the commissural gastric (CG) neuron (Robertson and Moulins, 1981; Simmers and Moulins, 1988)
directly excites several gastric mill CPG elements. A second projection
interneuron, the gastric inhibitor (GI) neuron, is identified in this
study for the first time and exerts conventional inhibitory effects on
several different gastric mill neurons. We show here that, in the
absence of spontaneous rhythmicity, activation of these two input
pathways, either separately or in combination, elicits distinct
patterns of gastric mill network activity that resemble different
rhythmic patterns observed in vivo. Importantly, the
integration of these extrinsic influences strongly depends on indirect
network-mediated interactions within the gastric mill circuit itself.
Parts of this work have been published in abstract form (Combes et al.,
1995).
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MATERIALS AND METHODS |
All experiments were performed on adult European rock lobsters,
Homarus gammarus, purchased from local commercial suppliers and kept in aquaria with fresh running seawater maintained at 16°C.
Before experiments, animals were cold anesthetized in ice for ~30
min. For in vitro experiments, the stomatogastric nervous system (STNS) was isolated from the foregut (Fig.
1A) as described by
Combes et al. (1993) and consisted of the stomatogastric ganglion (STG), bilateral commissural ganglia (CoG), the single oesophageal ganglion (OG), and their interconnecting nerves, including the single
stomatogastric nerve (stn) (Fig. 1B). Single motor
axon types were obtained by dissecting out distal gastric mill motor nerve branches to their corresponding muscles (Fig.
1A). The STNS was then transferred to a Petri dish
and superfused continuously with aerated artificial seawater maintained
at 15-18°C with a thermoelectric cooling system (Midland Ross Inc.).
The saline composition was (in mM): NaCl 479.12, KCl 12.74, CaCl2-2H2O 13.67, MgSO4 10, Na2SO4 3.91, and HEPES 5, buffered to pH
7.45.
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Figure 1.
STNS of the lobster Homarus
gammarus. A, Left lateral view of the foregut
showing the STNS in situ and gastric mill muscles
gm1-4, 6, and 9. B, STNS in vitro. The
somata of gastric mill motoneurons (GM, LG, MG, DG, and LPG) are
located in the STG, and their axons project caudally via the dorsal
ventricular nerve (dvn) into their respective bilateral
motor nerve roots. The two CoGs and the OG are connected to the STG via
the bilateral inferior oesophageal nerves (ions), the superior
oesophageal nerves (sons), and the single, mainly afferent stn.
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Extracellular recordings were made with platinum wire electrodes placed
against appropriate nerves and isolated electrically with Vaseline.
Intracellular recordings were made with glass microelectrodes placed in
the soma or the neuropile of neurons after desheathing the
corresponding ganglion. Microelectrodes (tip resistance of 10-30 M )
were filled with 3 M KCl for recording-stimulation or with
2.5 M LiCl and 3% Lucifer yellow (Sigma, Quentin
Fallavier, France) for labeling neurons. Dye was injected
iontophoretically with negative current (5-10 nA, 20 min) and
visualized with blue light illumination (450-490 nm). In some
experiments, intense illumination for 30 min or more was used to
photoinactivate injected neurons (Miller and Selverston, 1979).
Motoneurons were identified by correlating intrasomatic spikes with
action potentials recorded along terminal motor nerves branches (see
above). After impalement, the CG and GI projection neurons were
identified in each CoG by their axonal projections in the superior
oesophageal nerve (son) and the stn, and according to their different
postsynaptic effects on gastric mill motoneurons.
In several experiments, electromyographic recordings were made from
gastric mill muscles of freely behaving lobsters using EMG recording
methods as described previously (Clemens et al., 1998). Briefly,
Teflon-insulated silver wire electrodes (diameter of 125 µm) were
implanted into appropriate muscles via holes drilled in the
cephalothorax and connected via flexible wires to
laboratory-constructed extracellular amplifiers. Conventional
techniques for in vivo and in vitro experiments
were used for display, storage, and transcription of recorded data.
Labeling of the gastric motoneuron (GM) population was also made by
passive axonal migration of cobalt chloride (8.5% in distilled water)
from the cut end of the dorsal anterior gastric nerve (dagn) (Fig.
1B) toward the STG. Migration occurred for 36 hr at
4°C, and then the preparation was treated with 2% ammonium sulfide, fixed for 1 hr in 2.5% glutaraldehyde, dehydrated, and cleared in
methyl salicilate (Simmers and Moulins, 1988).
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RESULTS |
The gastric mill system of Homarus
Muscles responsible for rhythmic movements of the one medial and
two lateral gastric mill teeth in the lobster foregut and their
innervation by the STNS are illustrated in Figure 1A
(Maynard and Dando, 1974; Hartline and Maynard, 1975; Selverston and
Moulins, 1987). The medial tooth produces alternate phases of
protraction and retraction mediated by the gm1 and gm2 muscles
(innervated by GMs) and the gm4 muscle [innervated by the dorsal
gastric (DG) motoneuron], respectively. Alternate closing and opening
movements of the lateral teeth are driven by the gm6 and gm9
[innervated by lateral gastric (LG)-medial gastric (MG) motoneurons]
and gm3 [innervated by lateral posterior gastric (LPG) motoneurons]
muscles, respectively (Fig.
1A,B).
Spontaneous gastric mill output patterns
Typical spontaneous rhythmicity of the gastric mill network
in vitro is illustrated in Figure
2A. This version
of gastric mill motor output (which we designate "type I") consists
of MG and GM motoneurons, which innervate lateral and medial teeth
power-stroke muscles, firing synchronous bursts in antiphase with
motoneurons (Fig. 2A, LPG trace)
that innervate antagonistic return-stroke muscles.
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Figure 2.
Gastric mill output patterns observed in an
isolated STNS (A) and in vivo
(B). A, Extracellular recordings
of gastric mill motor nerves in vitro. GM motoneurons
(medial tooth protraction) fire in antiphase with LPG motoneurons
(lateral teeth opening; ellipse) and in phase with the
MG motoneuron (lateral teeth closure). Icons on the
right show teeth movements produced by each motoneuron
subgroup (P, protraction; O, opening;
C, closing). B, Electromyographic
recordings of gastric mill muscles in vivo showing two
distinct coordination patterns. B1, Pattern
similar to that seen in vitro (compare with
A) in which muscles innervated by the LPG and GM
motoneurons are alternately active (ellipse).
B2, Different animal in which the muscles
innervated by the LPG and GM motoneurons are coordinately active
(ellipse). In this pattern, protraction of the medial
tooth (GM neurons) occurs with lateral teeth opening (LPG
neurons).
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As seen in the EMG recordings from gastric mill muscles in Figure
2B1 (S. Clemens, personal
communication), the type I pattern is also expressed in
vivo (~80% of recorded animals). Again, the lateral teeth
opener muscle gm3 (innervated by the LPG neurons) is activated in
antiphase with the medial tooth protractor muscle gm2 (innervated by
the GM motoneurons). However, unlike in vitro, in the same
intact lobster, other types of gastric mill network activity are also
observed (Heinzel, 1988). One of these different versions, which we
refer to as "type II," is illustrated in Figure
2B2. In this sequence, which is spontaneously expressed <10% of the time, medial tooth protraction and lateral teeth opening (driven by GM and LPG motoneuron-innervated muscles, respectively) now occur conjointly (Figure
2B2, ellipse) and in
antiphase with lateral teeth closing (driven by the MG motoneuron).
Therefore, in vivo, a reorganization of gastric mill motor
output can occur in which lateral and medial teeth movements become
coordinated differently.
The gastric mill network of H. gammarus
In a first step toward exploring the neural basis of these
different gastric mill rhythms, we established the hitherto undescribed synaptic connectivity of the gastric mill motor network in H. gammarus. As seen in the pairwise intrasomatic recordings of
Figure 3A, like in other
crustaceans (Mulloney and Selverston, 1974a,b; Selverston and Moulins,
1987), inhibitory chemical synapses predominate in the H. gammarus gastric mill network. For example, the LG neuron inhibits
the LPG neurons, the DG neuron and interneuron 1 (Int1), and in each
case the short latency and 1:1 relationship between IPSP and
presynaptic impulse suggests a monosynaptic relationship. (Note
that for the two electrically coupled LPG neurons, because we did not
perform cell kills, we cannot tell whether one or both cells actually
receive direct LG neuron-mediated IPSPs). As in other Crustacea
(Selverston and Moulins, 1987), the single LG and MG motoneurons are
strongly electrically coupled and appear to behave as a functional
entity. Strong electrical coupling also exists between the GM and
LG-MG motoneurons (Fig. 3B; also see below). The 15 motoneurons [in addition to the solitary interneuron (Int1)] that
constitute the lobster gastric mill network are classified into four
subgroups; two of these subsets innervate the opener (two LPG
motoneurons) and closer muscles (LG and MG motoneurons) of the lateral
teeth subsystem, whereas the remaining two subgroups innervate the
protractor (GM motoneurons) and retractor (one DG motoneuron) muscles
of the medial tooth subsystem. Neurons within each functional group
(LG-MG, GM, and LPG neurons) are also electrically coupled.
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Figure 3.
Gastric mill network of H.
gammarus. A, Dual intrasomatic recordings that
reveal probable monosynaptic inhibitory connections. Each
panel shows superimposed oscilloscope sweeps triggered
by impulses in the presynaptic LG neuron. B, Synaptic
wiring diagram derived from recordings as in A.
Stick and ball symbol, Chemical inhibitory synapse;
resistor symbol, electrical coupling.
Numbers denote the number of neurons of each type.
Neurons of each functional group are electrically coupled, including
the LG-MG neurons. Tooth movement driven by each neuron type is also
indicated: O, C, opening and closing of
lateral teeth, respectively; P, R,
protraction and retraction of medial tooth, respectively.
C, Anatomical evidence for 10 GM motoneurons after
cobalt (CoCl2) backfill from their axons in the
dagn; 10 STG somata are labeled.
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Unlike spiny lobsters and crabs, which possess only four GM motoneurons
(Mulloney and Selverston, 1974a,b; Selverston and Moulins, 1987),
H. gammarus has 10 GM neurons. This is demonstrated in
Figure 3C, which shows a cobalt backfill of the dagn, which carries all of the GM neuron axons. In 4 of 4 preparations, 10 STG
somata were labeled. This anatomical finding was confirmed by
systematic intracellular recording from all STG neurons; in 3 of 3 preparations, 8-10 GM neurons could be identified electrophysiologically.
Gastric mill CPG flexibility: role of projection interneurons and
network connectivity
To understand how the gastric mill network can express the
distinctly different motor programs seen in vivo (Fig.
2B), we investigated inputs to the network that might
be responsible. More precisely, because in the type II pattern (Fig.
2B2), the LPG and GM motoneurons fire
together instead of in alternation as in the type I pattern (Fig.
2B1), we sought an extrinsic excitatory
influence that could force these two subgroups to fire in phase rather
than in antiphase.
CG interneuron
A source of excitatory input to the gastric mill network is the
pair of CG interneurons. First identified by Robertson and Moulins
(1981), the single CG neuron in each CoG projects its axon to the STG
via the ipsilateral son and the stn (Fig. 1). It was also shown
previously that in Homarus (Simmers and Moulins, 1988) each
CG neuron makes direct excitatory synapses onto the GM motoneurons. We
show here that the CG neurons also directly excite the LPG neurons. As
seen in Figure 4A, each
CG neuron spike is always followed at constant delay by EPSPs in
the GM and LPG neurons. The monosynapticity of these synaptic responses
is further suggested by their persistence in high
Ca2+-Mg2+ saline (data not
shown) (Simmers and Moulins, 1988). In contrast, no discrete
synaptic activity arising from the CG neuron was observed in LG-MG
neuron recordings (Fig. 4A).
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Figure 4.
CG interneuron interactions with the gastric mill
network. A, Superimposed sweeps showing CG
neuron-derived EPSPs in the LPG and GM neurons but not in the LG
neuron. Schema at top right shows the
synaptic relationship between the CG neuron and the gastric mill
network. B, Suprathreshold depolarization of the CG
neuron excites the GM and LG neurons but inhibits the LPG neuron
(left). However, when the LG neuron is continuously
hyperpolarized by intracellular current injection
(right), CG neuron stimulation now causes coordinated
LPG and GM neuron excitation. Calibration: vertical
bars, 10 mV; horizontal bar, 2 sec.
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To test whether the CG neuron could be responsible for the GM-LPG
neuron coactivation seen in the type II gastric mill pattern, the
interneuron was periodically stimulated with pulses of depolarizing current while recording from neurons of the different gastric mill
network subgroups. As seen in Figure 4B,
left, cyclic CG neuron firing activated the GM motoneuron
(third trace) but surprisingly inhibited the LPG
neurons (second trace). In contrast, the LG neuron,
which receives no direct input from the CG neuron (Fig. 4A), was also strongly excited during the activity of
the interneuron. The explanation for this apparent paradox lies
in the synaptic interactions within the gastric mill network itself.
Specifically, the LG-MG neuron inhibition of the LPG neuron overcomes
the CG neuron excitation. This opposing synaptic effect is revealed in Figure 4B, right, in which the LG neuron
was held continuously hyperpolarized and silent during CG neuron
stimulation. With the LG neuron (and the electrically coupled MG
neuron) thus functionally removed from the network, each burst evoked
in the CG interneuron now elicited synchronous spike trains in the LPG
and GM motoneurons (Fig. 4B, compare
right, left panels). Therefore,
hyperpolarization of the LG neuron released the LPG neuron from the LG
neuron inhibition and permitted it to fire in response to direct
excitation from the CG neuron.
Given that the LG-MG neuron group does not receive synaptic input from
the CG neuron, how is the LG-MG neuron group activated, and hence the
LPG neurons inhibited, by CG neuron activity? Here, again, the most
likely explanation is a network-mediated effect in which LG neuron
activation during CG neuron firing is caused by the strong GM-LG
neuron electrical coupling (Fig. 4, schema). Several
experimental arguments support this conclusion. First, excitation by
the CG neuron of all 10 GM motoneurons provides a powerful drive to the
LG-MG neurons. This combined electrotonic action of the GM neuron
ensemble can be seen in Figure 5 in which an LG neuron was recorded during current injection into one or two
simultaneously recorded GM neurons. Both depolarization
(top) and hyperpolarization (bottom) of the GM
neurons caused a corresponding depolarizing or hyperpolarizing LG
neuron response that was proportional to the number of manipulated GM
neurons (Fig. 5, compare left, right side
of each panel).
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Figure 5.
Electrical coupling between GM and LG motoneurons.
Depolarization of one (top left) or two (top
right) GM neurons by current injection (arrows)
depolarizes the LG neuron. Hyperpolarization of one (bottom
left) or two (bottom right) GM neurons by
current injection (arrows) hyperpolarizes the LG neuron.
Note the increased postsynaptic response when both GM neurons are
manipulated with current. Calibration: vertical bars, 20 mV; horizontal bar, 1 sec.
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A second direct argument that inhibition of the LPG neurons by the
gastric mill network (via the LG neuron) is stronger than the CG neuron
excitation derives from experiments (n = 4) in which the GM neurons were removed from the gastric mill network by
photoinactivation (Fig. 6). In the
preparation illustrated in Figure 6A, the gastric mill network was spontaneously arrhythmic under control conditions, and
CG neuron depolarization caused excitation and firing in an LG neuron
and inhibition of a tonically active LPG neuron (compare with Fig.
4B, left). Moreover, consistent with the
mixed direct and indirect effects of the interneuron on the LPG neuron,
the latter was initially excited (by direct CG neuron input; Fig. 6A, asterisk) when LG neuron firing was
still weak. [Note that the larger depolarizing transients in the
interneuron are EPSPs (Fig. 6A, bottom
insets), a feature of the CG interneuron (Robertson and
Moulins, 1981; Simmers and Moulins, 1988). The smaller depolarizing events are electrotonically decremented action potentials.] The entire
population of GM neurons was then backfilled into the ganglion with
Lucifer yellow placed in a Vaseline well built around their cut axons
in the dagn (Fig. 1B). After 12 hr of dye migration, the preparation was illuminated with blue light causing the
photo-ablation of the Lucifer-stained motoneurons (schema).
In this experimental situation, the CG neuron no longer excited the LG
neuron (because the GM neurons were dead), and hence CG neuron activity
was now able to directly excite the LPG neurons (Fig.
6B). That the silent LG neuron was still able to
influence the LPG neuron is shown in Figure 6C in which a
brief LG depolarization caused a transient LPG neuron inhibition during
CG neuron-evoked excitation. This experiment therefore strongly argues
that, although the LPG neuron receives direct excitation from the CG
interneurons, this effect is normally masked by inhibition from the
LG-MG neuron group, which is itself activated as a consequence of its
electrical coupling to the 10 CG neuron-activated GM neurons.
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Figure 6.
The CG interneuron excites the LG neuron via the
GM neurons. A, Stimulation of the CG neuron by
intracellular current injection excites the LG neuron and, after a
slight excitation (asterisk), inhibits an LPG neuron.
Note that the smaller depolarizing transients in the CG neuron are
action potentials, whereas the larger potentials are EPSPs
(insets are CG neuron recordings on faster time base).
B, After photoinactivation of Lucifer yellow-filled GM
neurons by intense blue light illumination, CG neuron depolarization no
longer activates the LG neuron and now excites the LPG neurons.
C, LG neuron activation by current injection during CG
neuron stimulation still causes LPG neuron inhibition. Calibration:
vertical bars, 10 mV; horizontal bars, 1 sec.
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Thus, two major conclusions arise from these results. First, despite
its direct excitatory connection with both the GM and LPG motoneurons,
activation of the projection CG neuron alone is unable to produce the
gastric mill pattern in which the LPG and GM neurons are coordinately
active (Fig. 2B2). Second, experiments with
current injection (Fig. 4B) and photoablation (Fig.
6B,C) suggest that such a network
configuration is feasible only if the LG-MG neurons are inhibited so
that excitation of the LPG neuron by the CG neuron can be expressed. We
present below evidence for a newly described projection neuron with
precisely such properties.
Commissural GI interneuron
The essential properties of this newly identified commissural
interneuron, which we have called the gastric inhibitor neuron because of its effects on the gastric mill network (see below), are
shown in Figures 7 and
8. The soma of the GI neuron lies
medially in each CoG, and its dendritic neuropile projects rostrally
into a region in which the CG interneuron is also located (Simmers and
Moulins, 1988) (Fig. 7A), although we have found no evidence of a synaptic connection between the two interneurons (Fig.
9A,B). Moreover, as with the two CG interneurons, the bilaterally paired GI
neurons do not appear to be coupled synaptically (data not shown). The
GI neurons project to the STG via the ipsilateral son and the stn (Fig.
7B,C). Stimulating a GI neuron with
intracellular current inhibits the LG, MG, and DG neurons (Fig.
8A), and superimposed oscilloscope sweeps (Fig.
8B) reveal that each GI neuron action potential
induces a constant latency IPSP (arrows) in all three neurons. These presumed direct synaptic influences of the GI neuron, which were not found on other gastric mill network neurons, are summarized in Figure 8, schema.
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Figure 7.
Identification of descending commissural GI
interneuron. A, Camera lucida drawing of a Lucifer
yellow-stained GI neuron in the right CoG; the GI neuron soma lies
between the emergence of the ion and son through which its axon runs
toward the stn. CG neuron soma position in the same CoG is also
indicated. B, Each GI neuron spike is correlated 1:1
with an axonal spike recorded extracellularly in the son and then in
the stn (5 superimposed oscilloscope sweeps). Calibration:
vertical bar, 5 mV; horizontal bar, 2 msec. C, Geometry of GI neurons in the STNS; their
somata are located one in each CoG and their axons project to the STG
via the son and the stn.
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Figure 8.
Synaptic relationship of the GI interneuron with
the gastric mill network. A, Depolarization of the GI
neuron inhibits the LG (top), MG
(middle), and DG (bottom) neurons.
B, Faster time base (5 sweeps in each recording). Each
GI neuron spike is correlated 1:1 with a constant latency IPSP
(arrows) in the MG, DG, and LG neurons. Diagram at
right summarizes these synaptic relations. Calibration:
vertical bars, A, 10 mV;
B, 5 mV; horizontal bars,
A, 2 sec; B, 10 msec.
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Figure 9.
Different gastric mill patterns encountered
in vivo can be reproduced in vitro by
activating the CG and GI interneurons. Schema summarizes
synaptic relationships between descending CG and GI neurons and the
gastric mill network. A, Periodic depolarization of a
tonically active CG neuron alone generates a gastric mill pattern that
resembles a type I pattern (LPG and GM neurons fire out of phase;
ellipse) seen spontaneously in vivo.
B, In the same preparation, rhythmic activation of the
GI neuron alone also produces a pattern in which LPG and GM neuron
bursts are in phase opposition. Here, however, the LPG neuron fires in
phase with the stimulated interneuron. C, Simultaneous
activation of the CG and GI interneurons organizes gastric mill
activity into a type II-like pattern, similar to that spontaneously
expressed in vivo; here, the LPG and GM neurons fire in
phase (ellipse) and in antiphase with the MG
neuron.
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Activation of the CG and GI projection neurons can reproduce types
I and II gastric mill output patterns
Finally, to examine and compare the actions of the excitatory CG
and inhibitory GI neurons on the gastric mill network, we stimulated
them separately and together in otherwise spontaneously arrhythmic
preparations. To assess their capacity to elaborate coordinated fictive
motor patterns similar to those generated spontaneously, the
interneurons were driven with cyclic depolarizations at periods similar
to those of gastric mill network rhythmicity. One of three such
experiments in which both interneurons and the gastric CPG neurons were
recorded simultaneously is illustrated in Figure 9, which also shows
the combined synaptic wiring diagram of the descending interneurons and
the motor network. First, rhythmic bursts evoked in the excitatory CG
neuron alone (Fig. 9A) immediately elicited coordinated
motor bursting similar to the type I gastric mill pattern that is
normally spontaneously expressed under in vitro conditions
(Fig. 2A). Thus, the MG and GM neuron bursts occurred
in phase with CG neuron activity and in antiphase with the LPG neurons
(ellipse). A pattern with a qualitatively similar phase
relationship was also produced by rhythmic stimulation of the GI neuron
alone (Fig. 9B). Here, however, unlike the response to CG
neuron activation, the MG and GM motoneurons fired in antiphase with
the GI neuron. In contrast, coactivation of the two descending interneurons induced a gastric output similar to the type II pattern spontaneously expressed in vivo (Fig. 2B);
the LPG and GM motoneurons now fired bursts conjointly and in time with
the interneurons but in antiphase with the MG neuron (Fig. 9, compare
C, ellipse, with A, B,
ellipses). In all cases, the evoked pattern persisted for as
long as the interneuron(s) was stimulated (data not shown). Therefore,
by selective or simultaneous activation of two parallel projection
pathways, replicas of distinct gastric mill motor patterns encountered
in vivo can be generated in vitro, even in the
absence of spontaneous network rhythmicity.
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DISCUSSION |
Functional flexibility of the gastric mill network
The purpose of this study was to explore the capability of a
central motor pattern-generating network to produce different activity
phenotypes seen in vivo. After determining the synaptic connectivity of the previously undescribed gastric mill CPG in the
stomatogastric ganglion of Homarus gammarus, we show that appropriate motor pattern selection can occur as a result of extrinsic input from two types of projection neurons arising from each of the
bilateral commissural ganglia. Thus, activation of the excitatory CG
interneuron alone can drive a type I fictive gastric mill motor pattern, whereas simultaneous stimulation of both the GI and CG neurons
immediately induces a type II-like gastric mill output pattern.
In the stomatogastric system of a number of decapod species, it is now
established that different motor patterns can be elicited from the STG
networks by activation of different projection neurons or the presence
of neuromodulatory substances (Hooper and Marder, 1984;
Harris-Warrick et al., 1992; Meyrand et al., 1994; Norris et al., 1994;
Marder and Calabrese, 1996; Blitz and Nusbaum, 1997). In virtually all
reported cases, however, the network reorganization appears to rely on
long-lasting neuromodulatory instruction involving relatively slow
changes in synaptic and cellular properties within the target CPG
itself (Getting and Dekin, 1985; Harris-Warrick et al., 1992; Morton
and Chiel, 1994). To date, the ability of multiple input pathways, such
as those mediating movement-related sensory feedback, to achieve rapid
network reconfiguration is much less well documented. This is in large
part attributable to the difficulty in identifying the ensemble of
pathways involved, as well as a lack of knowledge of the way in which
they impinge on their target network. The only other well characterized
example of proprioceptive input in the stomatogastric system is an
identified set of muscle receptors, the gastropyloric receptor (GPR)
cells (Katz et al., 1989). Interestingly, the GPR cells exert classical fast-acting synaptic influences on several STG neurons, although here
again, their primary action appears to be long-lasting neuromodulatory influences on intrinsic bursting properties (Katz and Harris-Warrick, 1989).
That the ongoing output of rhythmic neural networks can be modified by
activation of previously silent input neurons has been reported. For
example, in the locomotor systems of both the mollusc Clione
and Xenopus embryos, recruitment of input projection neurons causes faster and more vigorous swimming motor patterns (Arshavsky et
al., 1989; Sillar and Roberts, 1993). However, in both cases, the basic
temporal structure of the target motor program remains unchanged. In
contrast, recruitment of an identified projection neuron that selects a
different rhythmic motor program has already been reported in the
gastric mill system of the crab (Norris et al., 1994), and activation
of a single brain command neuron switches crab scaphognathite movements
from forward to reverse ventilatory motor patterns (DiCaprio, 1990).
Our present data reveal that different network coordinations can be
configured by separate or combined activation of dual input pathways to
the same motor circuitry in vitro and that the output
patterns produced closely resemble two distinct gastric mill motor
programs seen during spontaneous rhythmicity in vivo.
The gastric mill system in different species
The essential features of the Homarus gastric mill CPG
are similar to those described previously in other species, such as the
spiny lobsters Panulirus and Palinurus (Maynard
and Dando, 1974; Mulloney and Selverston, 1974a,b; Hartline and
Maynard, 1975) and crabs (Weimann et al., 1991; Katz and Tazaki, 1992). One notable difference, however, is in the number of motoneurons innervating the protractor (gm1) muscle of the gastric medial tooth.
Instead of only four GM motoneurons, as in Macrurans and Brachyurans, we find that the gm1 muscle of
Homarus is innervated by 10 excitatory motoneurons. This
large population of medial tooth power-stroke motoneurons contrasts
with the relatively small number of neurons making up the other
functional motoneuron subgroups: two for both the power-stroke (LG-MG
neurons) and return-stroke (LPG neurons) muscles of the lateral teeth
and only one (DG neuron) for the medial tooth return-stroke muscle. The
reason for this difference both within the lobster network and between
the gastric mill networks of different species is unknown, although our
results indicate that the large number of GM neurons in
Homarus plays a crucial role in biasing projection neuron
input within the gastric mill network itself (see below).
Other interspecies differences involve certain synaptic connections
between individual gastric mill network elements. For example, unlike
spiny lobsters (Selverston and Moulins, 1987), no evidence was found
for a rectifying electrical coupling between GM and LPG. Similarly, an
inhibitory synapse from LPG to LG reported in Panulirus
interruptus appears to be absent in the European lobster. Despite
these phylogenetic differences in synaptic wiring, gastric mill motor
output in vitro is remarkably similar in these species,
suggesting compensatory changes in cellular properties of individual
network neurons and/or that the above connections play a relatively
minor role in shaping the final pattern.
Direct versus indirect actions of projection pathways
Our results again demonstrate the importance of accounting for
indirect network effects in understanding precisely how an input
pathway operates (Hooper and Moulins, 1990). For example, although the
CG interneuron appears to monosynaptically excite the LPG motoneurons
of the gastric mill CPG, these neurons are in fact inhibited when the
CG neuron fires alone. This apparent paradox derives from the fact that
the CG neuron also has a strong inhibitory action on the LPG neuron via
a polysynaptic pathway mediated by the GM and LG-MG neurons (Fig. 6).
The predominance of the indirect CG to LG-MG neuron excitation is
undoubtedly assisted by the relatively large number (10) of GM
motoneurons, all of which appear to be directly excited by the
interneuron. In a manner equivalent to the convergence of visual
information in the vertebrate retina (Sterling et al., 1986), such
parallel information transfer via the GM motoneuron population is
likely to amplify the signal of the CG interneuron, thereby increasing
its influence on the LG-MG neurons. As a consequence, the CG
interneuron is able to induce a gastric mill motor output pattern in
which the medial tooth protractor GM motoneurons fire in antiphase with
the lateral teeth opener LPG motoneurons. It is only when the LG-MG
neurons are inhibited by the GI neuron that the CG neuron can induce
conjoint GM and LPG neuron activity.
The CG and GI interneurons are components of a sensory
feedback loop
Motor networks can express operational flexibility via a number of
different mechanisms (Getting and Dekin, 1985; Marder and Calabrese,
1996), including altering the way afferent information is conveyed to
the network. Employment of different input pathways to reconfigure
motor circuitry has been reported in several systems in both simple
vertebrates and invertebrates. For example, the switch between motor
patterns responsible for swimming and struggling in Xenopus
tadpole relies on changes in the recruitment of a population of sensory
interneurons (Soffe, 1991, 1997), and in the leech different reflex
responses to touch derive from sensory information processing in a
distributed population of intercalated interneurons (Lockery and
Kristan, 1990). The CG and GI neurons are also part of a sensory
feedback loop to the gastric mill CPG and are both postsynaptic to the
same identified primary mechanoreceptor neuron, the anterior gastric
receptor (AGR) (Simmers and Moulins, 1988; Combes et al., 1995, 1999).
Thus, the entire sensorimotor system comprises few elements (two CG and
GI interneuron pairs intercalated disynaptically between the single AGR
neuron and the 16 neuron gastric mill motor network), all of which are
accessible for electrophysiological recording and manipulation in
vitro. As shown in our accompanying paper (Combes et al., 1999),
AGR can activate the GI and CG interneurons in a selective manner,
similar to that reproduced by the direct interneuronal stimulation used
in the present study. That is, depending on its level of discharge, AGR
can activate the interneurons either singly or in combination, and thus
via the cellular and synaptic mechanisms identified here induce two
different gastric mill network configurations.
| |
FOOTNOTES |
Received Oct. 10, 1998; revised Feb. 9, 1999; accepted Feb. 12, 1999.
This work was supported in part by the Human Frontier Science Program.
Correspondence should be addressed to Denis Combes, Laboratoire de
Neurobiologie des Réseaux, Université Bordeaux I and Centre
National de la Recherche Scientifique, Unité Mixte de Recherche 5816, Avenue des Facultés, 33405 Talence, France.
| |
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TOP
ABSTRACT
INTRODUCTION
