Department of Biology, University of York, York YO10 5YW, United Kingdom
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ABSTRACT |
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Vehovszky, Ágnes and Christopher J. H. Elliott. Activation and Reconfiguration of Fictive Feeding by the OctopamineContaining Modulatory OC Interneurons in the Snail Lymnaea. J. Neurophysiol. 86: 792-808, 2001. We describe the role of the octopamine-containing OC interneurons in the buccal feeding system of Lymnaea stagnalis. OC neurons are swallowing phase interneurons receiving inhibitory inputs in the N1 and N2 phases, and excitatory inputs in the N3 phase of fictive feeding. Although the OC neurons do not always fire during feeding, the feeding rate is significantly (P < 0.001) higher when both SO and OC fire in each cycle than when only the SO fires. In 28% of silent preparations, a single stimulation of an OC interneuron evokes the feeding pattern. Repetitive stimulation of the OC interneuron increases the proportion of responsive preparations to 41%. The OC interneuron not only changes both the feeding rate and reconfigures the pattern. Depolarization of the OC interneurons increases the feeding rate and removes the B3 motor neuron from the firing sequence. Hyperpolarization slows it down (increasing the duration of N1 and N3 phases) and recruits the B3 motor neuron. OC interneurons form synaptic connections onto buccal motor neurons and interneurons but not onto the cerebral (cerebral giant cell) modulatory neurons. OC interneurons are electrically coupled to all N3 phase (B4, B4Cl, B8) feeding motor neurons. They form symmetrical connections with the N3p interneurons having dual electrical (excitatory) and chemical (inhibitory) components. OC interneurons evoke biphasic synaptic inputs on the protraction phase interneurons (SO, N1L, N1M), with a short inhibition followed by a longer lasting depolarization. N2d interneurons are hyperpolarized, while N2v interneurons are slowly depolarized and often fire a burst after OC stimulation. Most motor neurons also receive synaptic responses from the OC interneurons. Although OC and N3p interneurons are both swallowing phase interneurons, their synaptic contacts onto follower neurons are usually different (e.g., the B3 motor neurons are inhibited by OC, but excited by N3p interneurons). Repetitive stimulation of OC interneuron facilitates the excitatory component of the biphasic responses evoked on the SO, N1L, and N1M interneurons, but neither the N2 nor the N3 phase interneurons display a similar longer-lasting excitatory effect. OC interneurons are inhibited by all the buccal feeding interneurons, but excited by the serotonergic modulatory CGC neurons. We conclude that OC interneurons are a new kind of swallowing phase interneurons. Their connections with the buccal feeding interneurons can account for their modulatory effects on the feeding rhythm. As they contain octopamine, this is the first example in Lymnaea that monoaminergic modulation and reconfiguration are provided by an intrinsic member of the buccal feeding network.
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INTRODUCTION |
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Rhythmic behavior patterns are produced by a
system of central pattern generator (CPG) neurons, while modulatory
neurons can contribute by changing the rate of the rhythm, its pattern,
or the intensity of action potentials in the network (Katz and
Harris-Warrick 1990). The modulatory neurons may be classified
as extrinsic or intrinsic to the network (Cropper et al.
1987
; Katz 1995
; Katz and Frost
1996
). Extrinsic modulatory cells receive little feedback from
the network and can be situated in a different part of the nervous
system and act on multiple targets, e.g., central neurons and
peripheral muscles. Intrinsic modulatory neurons receive strong feedback from the CPG neurons and are usually rhythmically active themselves.
Snail feeding systems are productive models for the analysis of the
ways in which rhythmic patterns are generated and modulated (Benjamin and Elliott 1989; Kupfermann
1997
). The pond snail, Lymnaea stagnalis feeds
rhythmically using its radula, with three movements of approximately
equal duration: protraction of the radula, rasping across the food, and
then swallowing the food particles into the esophagus (Fig.
1A). Each feeding cycle takes about 3 s (Kemenes et al. 1986
;
Vehovszky et al. 1998
). A similar rhythmic pattern,
called fictive feeding, is produced by the isolated CNS, with rates of
up to 20 cycles/min. Many of the neurons that produce this pattern are
located in the buccal and cerebral ganglia (Fig. 1, B
and C). The buccal motor neurons (B1, B2 ··· B10),
CPG interneurons (N1, N2, N3), and modulatory interneurons (SO,
N1L) have been particularly well characterized (Benjamin and
Rose 1979
; Elliott and Benjamin 1985a
,b
;
Rose and Benjamin 1979
, 1981a
,b
; Yeoman et al. 1995
). The three kinds of N-cells fire in
turn, with the N1 interneurons firing in the protraction phase, the N2
during rasping (radula retraction), and the N3 during swallowing (Fig.
1B). Each of these three types of interneurons has been further subdivided into repeatably recognizable classes, each with
consistent anatomy and physiology, and their connections are well
described (Fig. 1B) (see Brierley et al.
1997a
,b
; Elliott and Benjamin 1985a
;
Yeoman et al. 1995
). Similar interneurons have been
found in other gastropods, e.g., Aplysia (Hurwitz et al. 1994
, 1997
) and Helisoma
(Quinlan and Murphy 1996
), and their connections have
been used to explain how the rhythm is produced (Brierley et al.
1997a
,b
; Elliott and Benjamin 1985a
,b
).
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Although many isolated CNS preparations of Lymnaea
show spontaneous fictive feeding, this can be enhanced in both strength and frequency by depolarizing a modulatory interneuron, SO, of which
there is only one between the two buccal ganglia (Elliott and
Benjamin 1985b). The SO can also initiate the feeding rhythm in
a quiescent preparation, although there are statistically significant differences between the spontaneous and SO-driven patterns
(Elliott and Andrew 1991
). Similar activation of feeding
can be achieved by depolarizing the paired N1L interneurons in the
buccal ganglia (Yeoman et al. 1995
), or the CV1
interneurons in the cerebral ganglia (McCrohan 1984
;
McCrohan and Kyriakides 1989
). Another cerebral
interneuron, the CBWC, has a weaker activating effect (McCrohan and Croll 1997
), while the serotonergic
cerebral giant cells (CGCs) modulate the rate and strength of the
rhythm, but cannot normally initiate a pattern in a quiescent
preparation (McCrohan and Audesirk 1987
; Yeoman
et al. 1994a
,b
, 1996
).
The buccal ganglia of Lymnaea stagnalis have a high
octopamine content (Hiripi et al. 1998), which was
accounted for by just three octopamine immunoreactive neurons called
the OC interneurons (Elekes et al. 1993
,
1996
; Vehovszky et al. 1998
). OC
interneurons display rhythmic activity pattern during both spontaneous
and interneuron-driven fictive feeding. They fire in the third,
swallowing (N3) phase of the feeding cycle and form synaptic
connections with the feeding neurons of the buccal ganglia, suggesting
that the OC interneurons are members of the feeding system
(Vehovszky and Elliott 2000
; Vehovszky et al.
1998
). Their octopaminergic nature is confirmed by
pharmacological experiments, as their synaptic connections to the B3
and N3p neurons are blocked by those octopamine antagonists that
inhibited feeding in intact animals (Vehovszky et al.
1998
, 2000
).
Here we show that the OC interneurons modulate all the other
interneurons and motor neurons of the buccal feeding network. We also
report the diversity of monophasic, biphasic, and long-term effects of
OC interneurons on the buccal feeding interneurons. These connections
provide the mechanisms by which the OC interneurons reconfigure and
modulate the activity of the buccal feeding network. The octopamine
content and the octopaminergic output connections of the OC
interneurons have already been established (Vehovszky et al.
1998, 2000
). Thus this is the first example in
Lymnaea when aminergic modulation comes from an intrinsic
member of the feeding system.
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METHODS |
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Animals
Adult pond snails (Lymnaea stagnalis) were obtained
from a dealer (Blades Biological, Kent, UK) kept in standard
snail water (Thomas et al. 1975) and fed on lettuce ad libitum.
Experiments on the isolated CNS
The CNS, including the buccal ganglia and a short length of
esophagus, was isolated and the connective tissue digested for 5 min
using a dilute (approximately 0.1%) solution of protease (Sigma type
XIV) in standard Lymnaea saline (Table
1). Glass capillary electrodes were
filled with potassium acetate and used to impale buccal neurons. The
large cells were identified visually; the smaller ones by their
activity pattern and synaptic relationships with the visually
identifiable cells (Benjamin and Elliott 1989; Brierley et al. 1997a
,b
; Yeoman et al.
1995
). The octopamine-containing OC interneurons described in
detail here are on the dorsal surface of the buccal ganglia near the
buccal commissure (Fig. 1) and are the only cells in this part of the
buccal ganglia that have electrical connections with the B4 motor
neurons (Vehovszky et al. 1998
).
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Experiments to identify buccal feeding neurons and to
characterize the effects of the OC interneurons on the feeding pattern were performed in normal Lymnaea saline (Table 1). To
characterize the individual connections the continuous perfusion of
standard Lymnaea saline was switched to the saline with
raised calcium and magnesium (Hi-Di saline, Table 1). The Hi-Di saline
raises the action potential threshold (Berry and Pentreath
1976; Elliott and Benjamin 1989
) and so reduces
the spontaneous activity of neurons and the effect of polysynaptic
pathways activated by intracellular stimulation of presynaptic neurons.
To separate the electrical and chemical connections, a high Mg/low Ca
solution was used (Table 1), which blocks the chemical but not the
electrical transmission between neurons (Elliott et al.
1992
).
In the experiments with the CGCs, we checked that dissection had not
overstretched the cerebro-buccal connectives. These nerves contain the
axons of left and right CGCs, which meet in the buccal ganglia with
mutually excitatory synapses with each other and form excitatory
synaptic connections with the buccal B1 motor neurons (McCrohan
and Benjamin 1980a,b
). The synchronous firing of CGC neurons,
their electrical coupling, and their excitatory effect to the buccal B1
neurons showed that the cerebro-buccal connectives were functional
during the experiments.
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RESULTS |
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OC interneurons in the feeding rhythm
OC NEURONS ARE ACTIVE IN THE SWALLOWING PHASE OF THE FEEDING CYCLE.
The OC interneurons display the same pattern of synaptic inputs and
firing activity as the other N3 phase neurons of the buccal feeding
system (Fig. 2). The OC interneurons are
inhibited in the first (N1, protraction) and second (N2, rasp) phases
of the feeding pattern and receive excitatory inputs during the N3
(swallowing) phase of the feeding cycle like the B4 motor neurons (Fig.
2, A, C, and E) or N3p interneurons (Fig.
2B). After intracellular activation of the
pattern-generating protraction phase SO (Fig. 2, A and
C) or N1L (Fig. 2B) interneurons, the feeding
pattern gradually builds up until the OC interneurons fire in bursts
during the third, swallowing phase of the feeding cycle (Fig. 2,
A-C). The intensity of OC firing is from 5 to 22 Hz,
depending on the preparation (Fig. 2D1) (Elliott and
Vehovszky 2000), and varies only slightly within each
preparation. The frequency of action potentials in the OC interneuron
is approximately the same as that of the B4 swallowing phase motor
neurons, (Fig. 2, A, C, and E). One-to-one firing
of action potentials is not usually seen, although during the N3 phase,
an OC interneuron may show small excitatory postsynaptic potentials
(EPSPs) that correspond to the action potentials in the B4 motor neuron
(Fig. 2C, small arrows, cf. Fig. 8D) (see also
Fig. 1 of Vehovszky and Elliott 2000
).
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OC NEURON STIMULATION EVOKES FICTIVE FEEDING.
Intracellular stimulation of OC neurons may also evoke fictive feeding
in silent preparations. In 23 of 82 (28%) preparations, a single short
depolarizing pulse to the OC interneuron activates fictive feeding
(Fig. 2E). As we can only impale one of the three OC
interneurons, we applied a relatively strong (25-35 Hz) burst for
2 s to the OC interneuron. This depolarizes the SO interneuron, and as the SO starts to fire faster, both the B4 neuron and the OC
interneuron are recruited to the fictive feeding pattern: strong N2
(rasp) inputs occur, as shown by the rapid, synchronized
hyperpolarization of the B4 motor neuron and OC interneuron (marked by
asterisks on Fig. 2E). On those preparations when a
single pulse to the OC fails to activate feeding pattern, repeated
current pulses injected into the OC neurons are more likely to evoke
the fictive feeding pattern (12 preparations of 29 tested, 41%, Fig.
2F). A series of 2-s stimuli, separated by 1-s
intervals, was applied to the OC interneuron for 10-15 s. Such a
pattern of stimulation mimics the rhythm of feeding and has previously
been shown to be more effective than tonic stimulation of the OC
(Elliott and Vehovszky 2000). This pulsed stimulation
gradually leads to the feeding pattern in both motor neurons and
interneurons (B1 and N1M in Fig. 2F) and, after four
cycles, recruits further OC activity as well. Again, the rhythmic
pattern occurs after and lasts longer that the actual duration of the
OC stimuli (Fig. 2F). The activation of feeding
neurons, moreover, does not require continuous firing of the OC
interneuron, as the depolarization and tonic firing activity on SO and
B4 neurons (Fig. 2E) or B1 and N1M neurons (Fig.
2F) is maintained after the end of OC stimulation.
OC ACTIVITY RECONFIGURES THE FEEDING PATTERN.
We demonstrated reconfiguration of the network by injecting
either depolarizing or hyperpolarizing currents tonically into the OC
interneuron, while driving fictive feeding with steady current
injection into the SO. When the OC is depolarized (Fig. 3A), the feeding pattern is
changed by the removal of the bursting activity in the B3 motor
neurons, which project in the dorso- and latero-buccal nerves, and are
thought to modulate the buccal glands (Benjamin et al.
1979; Carriker 1946
). In the hyperpolarizing experiment, the removal of the OC was correlated with a depolarization of the B3 motor neuron and its recruitment into the feeding rhythm (Fig. 3B1, section b).
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Connections from the OC interneurons to the buccal feeding neurons
To account for the firing pattern of the OC neurons, their ability to stimulate feeding rhythm, and the reconfiguration of the firing pattern, we have examined the connections from the OC interneurons to the other buccal feeding neurons in detail. The connections reported below were all monitored in Hi-Di saline, which elevates the firing threshold of the neurons and reduces the spontaneous activity and the polysynaptic effects evoked by presynaptic (OC) stimulation (see METHODS). Therefore our results suggest direct connections between OC and its followers.
SYMMETRICAL DUAL CONNECTIONS BETWEEN OC AND N3P
INTERNEURONS, BOTH CONTAINING ELECTRICAL (EXCITATORY) AND CHEMICAL
(INHIBITORY) COMPONENTS.
The N3p interneurons are, like the OC interneurons themselves, a type
of interneuron that receives excitatory inputs or fires in the
swallowing phase (Elliott and Benjamin 1985a). The N3p and OC interneurons are electrically coupled to each other with symmetrical connections (n = 36, Fig.
4, A and B). When
short (0.5-1 s) negative current is injected into either of these
interneurons, the other cell is hyperpolarized, while with depolarizing
currents, a symmetrical depolarization, accompanied by electrically
transmitted action potentials is seen in the other interneuron (Fig. 4,
A1 and B1). The connection is, however, very
rarely strong enough to evoke 1:1 spikes, unless both cells receive a
simultaneous depolarizing input. Furthermore, this is in fact a dual
connection with the electrical connection supplemented by a slower
chemical component. After suprathreshold OC stimulation, the N3p
returns to rest slowly from a more negative potential than it began,
indicating an additional hyperpolarizing effect after the electrical
response to OC stimulation (n = 35, Fig.
4A2). This is most obvious in the Hi-Di solution in which
both the electrical (excitatory) and chemical (inhibitory) components
of the responses are clearly visible (Fig. 4, A2 and
A3). Similarly, N3p stimulation evokes a complex response on
OC interneurons (n = 34, Fig. 4B) when the electrical response is combined with a hyperpolarization of the OC
interneuron. In both OC
N3 and N3
OC directions, 1:1 electrical PSPs can be seen (Fig. 4, A3 and B3), while
individual chemical PSPs cannot be resolved. The relative size of the
two components (chemical and electrical) of the OC 171 N3p connection
depends on the membrane potential level of the target neuron. When the N3p interneuron is deeply hyperpolarized (below
85 mV), the OC neuron
produces mainly a depolarizing effect (due to the electrical coupling,
Fig. 4C1). As the postsynaptic N3p cell is depolarized, the
inhibitory (chemical) component of the OC
N3p connection becomes
dominant over the excitatory (electrical) response (Fig. 4,
C2 and C3). This change in the relative
importance of the electrical and chemical components also happens when
the N3p interneuron is stimulated and the OC interneuron membrane
potential changes (not shown).
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OC INTERNEURONS FORM BIPHASIC (INHIBITORY FOLLOWED BY
EXCITATORY) CHEMICAL CONNECTIONS WITH PROTRACTION PHASE FEEDING
INTERNEURONS.
When an OC interneuron is stimulated, a biphasic synaptic input
(hyperpolarization followed by slower excitatory response) is seen in
the protraction phase SO, N1L, N1M interneurons (SO, n = 19; N1L, n = 13; N1M, n = 12; Fig.
5, A-C). This second,
depolarizing component will elicit action potentials if the starting
membrane potential is close to the threshold of action potential of the SO or N1L postsynaptic cells (Fig. 5A2). When the membrane
potential of the postsynaptic interneuron lies between 70 and
60
mV, the small initial hyperpolarization is not always visible, while
the following slow depolarization is more obvious (Fig. 5, A1,
B1, and C1). However, when the protraction phase
interneuron is already firing, the inhibitory component dominates,
which stops firing the follower interneuron (Fig. 5, B2 and
C2). As soon as the OC stimulation (OC burst) is over, the
follower cell resumes its firing activity (Fig. 5, B2 and
C2).
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OC NEURONS FORM EXCITATORY CONNECTIONS WITH THE VENTRAL
N2 INTERNEURONS BUT INHIBIT THE DORSAL N2 INTERNEURONS.
The OC interneuron excites some types of N2 interneurons, namely the
N2v interneurons located on the ventral surface of the buccal ganglia
(Brierley et al. 1997a,b
). OC stimulation is followed by
rapid bursts of the N2v neurons, with short latency and rapid decay
(n = 5, Fig.
6A1). After long perfusion in
Hi-Di saline, however, when the threshold of action potential
generation is higher, and the frequency of the action potentials in the
OC bursts is decreased, the synaptically evoked depolarization is
clearly separated from the endogenous bursts triggered on N2v
interneurons (Fig. 6A2).
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OC INTERNEURONS HAVE DUAL CHEMICAL AND ELECTRICAL
EXCITATION WITH THE N3T INTERNEURONS.
The connection of the OC interneurons with the tonically firing N3t
interneurons (Elliott and Benjamin 1985a) has also been examined in five preparations. Injecting currents of either polarity into the OC interneuron produces a small response on the N3t
interneuron with the same sign and duration, suggesting an electrical
connection (Fig. 6C1). When suprathreshold currents were
applied to the OC, a much larger response is seen in the N3t
interneuron, complete with action potentials (Fig. 6C2). The
increased response suggests that there is also a chemical excitatory
connection from the OC
N3t interneuron.
SYNAPTIC CONNECTION OF OC INTERNEURONS WITH THE FEEDING MOTOR NEURONS. The synaptic inputs evoked by OC neurons on most motor neurons correspond to the synaptic responses of the interneurons with which they are simultaneously active during feeding.
OC interneurons have electrical connections with all (B4, B4cluster, B8) motor neurons that fire in the same phase of the fictive feeding (Fig. 7A), although the coupling is the strongest between OC interneurons and B4 motor neurons (Fig. 7A1). Again, the electrical response between an OC and these follower neurons is very rarely large enough to evoke full sized action potentials. Moreover (contrary to the OC
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POSTSYNAPTIC EFFECTS OF OC STIMULATION IS DISSIMILAR TO
STIMULATION OF THE ELECTRICALLY COUPLED N3P INTERNEURON.
Both OC and N3p neurons are excited or fire in the swallowing phase of
feeding and receive simultaneous inhibitory inputs during N1 and N3
phases (Fig. 2B) (c.f. Elliott and Benjamin
1985a), which indicates that both the OC interneurons and the
N3p interneurons are included in the N3 (swallowing phase)
interneurons. However, they still may have separate roles in
feeding, if their connections to other members of the feeding network
are different. Therefore, in the next series of experiments, we
compared the effects of either OC or N3p interneurons on their
followers recording simultaneously from both OC and N3p interneurons
together with other buccal neurons.
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REPETITIVE STIMULATION OF OC FACILITATES THE EXCITATORY COMPONENT OF THE BIPHASIC SYNAPTIC INPUTS ON PROTRACTION PHASE INTERNEURONS BUT NOT THE DEPOLARIZATION OF N2V NEURONS. As we have already demonstrated (Fig. 2F), repetitive OC stimulation is more effective at evoking fictive feeding than a single stimulus. One reason for this may be that the biphasic postsynaptic effects received on protraction phase feeding interneurons after OC stimulation (Fig. 5) undergo homosynaptic facilitation (Figs. 9 and 10).
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Synaptic inputs received by OC interneurons from buccal feeding interneurons
The OC interneurons receive inhibitory inputs from most of the buccal interneurons. Stimulation of either an SO or N1L interneuron inhibits OC activity (14 of 19 on SO, 10 of 13 on N1L, Fig. 11, A and B), but single inhibitory postsynaptic potentials (IPSPs) are only occasionally seen on the OC records after each SO or N1L presynaptic action potential (not shown). Although OC neurons receive inhibitory inputs when N1M interneurons fire in N1 phase of feeding, intracellular stimulation of N1M neurons usually has much less effect on OC activity; with inhibitory response in only about one-half of the Hi-Di experiments (3 of 7, Fig. 11C) and single IPSPs are not visible.
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After stimulation of the N2d interneurons, the inputs on the OC interneurons are rather variable; inhibitory inputs were recorded only on one-half (5 of 10) of the preparations after N2d stimulation. Because of the wide range of latencies between the N2d stimulation and OC response even on the same preparation (Fig. 11, D1 and D2), direct connections from N2d interneurons to OC interneurons are not likely.
In the case of the N2v OC connections, however, N2v interneurons
reliably evoked typical N2 inputs on OC neurons, which are biphasic; a
short inhibition is followed by a depolarizing response (8 of 9, Fig.
11E).
The electrical coupling between OC interneurons and N3t interneurons
means that either hyperpolarizing or depolarizing N3t interneurons with
short pulses should evoke a weak electrical response on the OC
interneuron (Fig. 11F1). However, stimulating the
N3t even with longer depolarizing pulses has only a very small effect
on the OC (Fig. 11F2). Thus the connection between the OC and N3t neurons seems to be rather asymmetrical, as the N3t OC
connection is much weaker (Fig. 11F) than the OC
N3t excitatory input (Fig. 6C2).
As we mentioned earlier, N3p interneurons form complex, biphasic (excitatory/inhibitory) synaptic connection with OC neurons, the former being electrical, while the second is a chemically transmitted synaptic response (Fig. 4).
Connection between the OC interneurons and CGCs
We have also looked for connections from the OC interneurons to the serotonergic CGCs, which are feeding interneurons in the cerebral ganglia. The CGCs have no response to either single pulse (Fig. 12A) or repetitive OC stimulation (Fig. 12B) in any of the experiments (neither in normal solution nor in Hi-Di saline, n = 10), even though other buccal neurons (B3 motor neuron, Fig. 12A; B4 motor neuron, Fig. 12B) show their normal responses (OC stimulation, Figs. 7D2 and 8, B1 and D1).
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The CGC cells (on the same preparations), however, evoke excitatory
responses on OC neurons as well as on feeding motor neurons (Fig. 12,
C and D, n = 12), at which the
connections are already known (McCrohan and Benjamin
1980b). This confirms that the failure of OC
CGC response
is not caused by the lack of connections between the buccal and
cerebral ganglia. The spontaneous spikes recorded on CGC are often
followed by clearly visible 1:1 excitatory potentials on OC
interneurons (Fig. 12E), suggesting that CGC neurons have
direct connection with the OC interneurons.
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DISCUSSION |
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The data in this paper show that the octopamine immunoreactive OC interneurons can stimulate (Fig. 2) and reconfigure (Fig. 3) the Lymnaea feeding system. The OC interneurons also connect to the feeding interneurons and motor neurons (Figs. 4-9, 12, and 13). Here we review the connections and show that they can explain the ability of the OC interneuron to modulate the feeding pattern.
Overview of the synaptic connections formed by OC interneurons
OC interneurons form a wide variety of connections with buccal
feeding neurons, which include electrical, chemical, and mixed synapses
(Fig. 13). All responses have
approximately the same (200-400 ms) latency but different amplitudes
and durations (Fig. 13, B and C). Although we did
not record single chemical postsynaptic potentials on follower neurons
after OC stimulation, all the OC output connections described here
persist after 30-60 min of Hi-Di solution, which reduces the effect of
polysynaptic connections activated by presynaptic OC stimulation
(Berry and Pentreath 1976; Elliott and Benjamin
1989
) (see METHODS). Moreover, the OC output effects could not be explained by an interaction passing through any
previously known feeding interneuron, as the pattern of inputs recorded
in the buccal neurons following OC stimulation is different from that
seen when stimulating any one of the N1, N2, or N3 interneurons (Brierley et al. 1997a
,b
; Elliott and Benjamin
1985a
,b
; Rose and Benjamin 1981a
,b
;
Vehovszky and Elliott 1995
; Yeoman et al.
1995
) or OM mechanoreceptor neurons (Elliott
and Benjamin 1989
). The latency of OC responses, however, is
much longer than that of cholinergic chemical postsynaptic potentials
in the Lymnaea buccal ganglia (Elliott and Benjamin
1985b
; Elliott and Kemenes 1992
; Vehovszky and Elliott 1995
), and this suggests that the
OC interneurons may use second-messenger pathways for their effects.
Octopaminergic modulation coupled to adenylate cyclase is common in
insects (Bounias 1987
; Chyb et al. 1999
;
Nathanson and Greengard 1973
; Orchard et al.
1983
; Robb et al. 1994
), and some reports
suggest that a similar mechanism may be present in molluscan systems
(Capasso et al. 1991
; Chang et al. 2000
;
Gerhardt et al. 1997
).
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The strongest, short-term excitatory connection formed by the OC
interneurons is electrical coupling with the N3 phase neurons (B4,
B4cl, B8 motor neurons, N3p interneurons, Fig. 13A,
traces 1 and 4). The electrical coupling of N3t
interneurons and OC neurons is much weaker, but the N3t interneurons
receive an additional, rather strong excitatory effect from the OC
interneurons (Figs. 6C2 and 13, trace 2). The
other excitatory output is the chemically transmitted depolarizing
effect on the N2v interneurons, which often triggers a short, quickly
terminating burst in the N2v interneuron (Fig. 13A, trace
2), due to its endogenous plateauing property (Brierley et
al. 1997a).
On the N3p interneurons, the short (electrical) excitatory inputs are followed by a longer lasting (chemical) inhibitory effect (Fig. 13A, trace 4). These OC 171 N3p connections are symmetrical, as OC interneurons receive similar biphasic (electrical excitatory/chemical inhibitory) input from N3p interneurons (Fig. 4). The synaptic response recorded on protraction phase (SO, N1L, N1M) interneurons is a short inhibitory response followed by a second, longer-lasting excitatory component (Fig. 13A, trace 3). Both responses are chemical ones. The N2d phase interneurons receive a short latency but rather long-lasting hyperpolarization from OC interneurons (Fig. 13A, trace 5).
The synaptic inputs that the motor neurons receive from the OC interneurons are generally similar to those received by the interneurons that fire in the same phase of feeding cycle. For example, N3 phase (B4, B4Cl, and B8) motor neurons like N3p interneurons are electrically coupled to OC interneurons, while N2 phase (B2, B3, B10) motor neurons, like N2d interneurons receive hyperpolarizing inputs. The N1 phase B7 motor neurons have similar biphasic (inhibitory than excitatory) synaptic inputs to the SO, N1L, and N1M interneurons. The exceptions are the B1 and B6 protraction phase motor neurons, which receive only excitatory inputs without any inhibitory component.
Although OC interneurons receive excitation from the cerebral CGC
neurons, a reverse connection was not found. This confirms the previous
suggestion based on morphological results (Vehovszky et al.
1998) that the OC interneurons are local interneurons of the
buccal feeding system.
Biphasic outputs of OC interneurons provide activity-dependent effects on the feeding network
A notable characteristic of the OC outputs is that the response is often biphasic (Fig. 13, A and B). This may be achieved by a combination of chemical synapses (inhibition followed by excitation), which is seen on both motor neurons (B5, B7) and interneurons (SO, N1L, N1M) or by the dual (excitatory) electrical then (inhibitory) chemical connection onto the N3p interneurons. This means that the functional consequence of spikes in the OC interneuron depends on the membrane potential of the follower cell. In other words, the OC interneurons will have an activity-dependent effect on the feeding network. First, when the whole feeding system is quiet, the membrane potentials of the follower cells will be near the resting value and any inhibitory inputs produced by stimulating the OC interneurons will be small. Thus the dominant effect will be excitation, resulting from the excitatory components that are chemical onto the N1 interneurons and electrical onto the N3p interneurons. However, during fictive feeding, the membrane potential of the follower neurons is generally more depolarized. Now, the main synaptic response after OC bursts is a brief inhibition of the protraction and the N3p swallowing phase interneurons. This shortens both the N1 and the N3 phases, so that OC firing in the swallowing phase increases the feeding rate (Fig. 3, B2 and B3). The sharp excitatory response on N3t interneurons does not seem to affect the duration of the swallowing phase perhaps because the inhibitory input soon takes over.
Interneurons with biphasic inputs have been described in other central
pattern generators of Lymnaea (Benjamin and Winlow 1981; Skingsley et al. 1993
), in the buccal
feeding system of Aplysia (Fiore and Gepetti
1981
; Gardner 1977
), and in the swimming network
in Tritonia (Getting 1981
,
1983
). However, in the Lymnaea feeding system
this is the first example where the functional role of the biphasic
inputs formed by a modulatory interneuron is suggested.
Longer lasting effect of OC connections modulates the feeding system
The wide range of response durations is another especially interesting aspect of the connections formed by OC interneurons (Fig. 13C). The short-term effects (intraphasic modulation) last during the presynaptic OC bursts (marked by dotted lines in Fig. 13, A and C), but other outputs have their main effect after the spiking activity (burst) of the OC interneuron is over.
With its strong, short-term excitatory connections with other N3 phase
neurons (electrical coupling with both the N3p interneurons and with
the N3t interneurons, supplemented by the chemical excitatory effect on
the N3t interneurons), the OC interneurons facilitate the overall
firing activity of all the N3 neurons, increasing the outputs to the
feeding muscles contracting in swallowing phase. This reflects a
general feature of the feeding system as neurons firing in the same
phase are electrically coupled (Staras et al. 1998;
Yeoman et al. 1995
). The short inhibitory effects of OC interneurons on protraction phase neurons, however, prevents them from
firing (and the protraction muscles to contract) simultaneously in the
swallowing phase.
The longer term outputs are delayed with respect to the burst of
the presynaptic OC neuron. After a short inhibitory response recorded
in the N1 (protraction) phase interneurons during the OC burst, the
following excitatory effect lasts for up to 7-8 s (Fig.
13C), far above the duration of an average feeding cycle (this is 3 s corresponding to the top 20 bites/minute feeding rate
of intact animals) (Kemenes et al. 1986;
Vehovszky et al. 1998
). This fits well with the feeding
pattern in which protraction phase neurons are inhibited during
swallowing phase, but their membrane can be excited later as the next
feeding cycle follows after swallowing. This polycyclic excitatory
effect produced by OC neurons seems to be a significant difference
between the OC and previously described modulatory interneurons in the
Lymnaea feeding system is that these cells (CV1, N1L, and
SO) mostly drive feeding as long as the stimulus is maintained
(Elliott and Benjamin 1985a
,b
; McCrohan
1984
; McCrohan and Croll 1997
; Yeoman et
al. 1995
) while the effect of the OC interneuron tends to drive
fictive feeding once the stimulus is ended (see Figs. 2, D
and E, and 10B) therefore promotes the feeding pattern.
The reciprocal biphasic effects between OC and N3p neurons (a
short electrical excitatory connection followed by a longer inhibitory
chemical one) seem paradoxical first, as both neurons fire in the same
N3 phase of feeding. One explanation is that the inhibitory component
of the OC connections helps to terminate the N3p bursts (previously
facilitated by the short-term electrical coupling). This shortens the
feeding cycles and increases the feeding rate (as seen in Fig. 3),
therefore the reciprocal OC and N3 inhibitory connections contribute to
the pattern generation. Although all previous work in
Lymnaea has suggested that the groups of neurons firing in
the same phase have mutually (electrical) excitatory effects
(Staras et al. 1998; Yeoman et al. 1995
),
the OC 171 N3p relationship has a similarity to the Aplysia
feeding system where the histaminergic B52 neurons fire in the same
phase and have reciprocal inhibition, although the B52 neurons are not electrically coupled (Evans et al. 1999
). Reciprocal
inhibition is suggested to have a central role in the mechanisms of
pattern generation (see review by Cropper and Weiss
1996
).
The longest duration effects of the OC interneuron are inhibition of
the B2 and B3 motor neurons (Figs. 7, D1 and D2,
8, A and B, and 13C).
Morphological data (Benjamin et al. 1979; Perry et al. 1998
) suggest that these giant neurons innervate of the esophagus. The peristaltic activity of the esophagus may not
necessarily be linked tightly to the individual feeding phases produced
by the buccal musculature (Perry et al. 1998
).
Different outputs of OC and N3p interneurons allow reconfiguration of the feeding network
Although the pattern of inputs and firing activity of
OC and N3p interneurons are almost identical to inhibitory N1
and N2 inputs and excitatory inputs during N3 phase (Fig.
2B), they evoke different inputs on (B3, B5, B4) motor
neurons (Fig. 8). This indicates that OC and N3p interneurons represent
functionally different subgroups in the populations of N3 phase
interneurons. The physiological differences between OC and N3p
interneurons are reflected by earlier morphological data, as the axonal
branching patterns of OC interneurons and N3p neurons are completely
different from each other (Elliott and Benjamin 1985a;
Vehovszky et al. 1998
). Pharmacological differences are
also present, as only OC interneurons contain and use octopamine
(Vehovszky et al. 1998
, 2000
); the
transmitter for N3p neurons is not established yet. Furthermore, the OC
and N3p interneurons will reconfigure the network in different ways,
with (for example) recruitment of the OC interneurons reducing the
activity of the B3 motor neuron, and recruitment of N3p interneurons
increasing the activity of the B3 motor neurons. In addition, the OC
interneurons, but not the N3p interneurons, can enhance the overall
fictive feeding rate, with the maximal rate (20 cycles/min) occurring
only when both the OC and SO fire.
Previous analyses of the rhythmic pattern in Lymnaea
demonstrated that modulatory interneurons affect the feeding intensity (motor neuron firing rate; CGC) (McCrohan and Audesirk
1987; Yeoman et al. 1996
) or rhythmic rate (SO,
N1L, CGC, CV1) (Elliott and Benjamin 1985b
;
McCrohan 1984
; McCrohan and Kyriakides
1989
; Yeoman et al. 1996
). Additionally the SO
and CGC interneurons alter the relative proportion of time spent in
each feeding phase (Elliott and Andrew 1991
;
Yeoman et al. 1996
). OC interneurons do the same, as the
length of N1 and N3 phases decrease during OC activity (see Fig.
3B3). Additionally, OC neurons also change the pattern of
motor outputs (due to the differential firing patterns of the motor
neurons) during its activity. In Helisoma buccal system a
similar reconfiguration can be achieved by hyperpolarizing the swallowing phase N3a interneurons, which seems to have many homologous physiological features with the Lymnaea OC interneurons
(Quinlan and Murphy 1996
).
Electrically coupled modulatory neurons that use different
transmitters and are able to reconfigure the network in different ways
can also be found in crustacean pattern generators (Eisen and
Marder 1984; Marder and Eisen 1984
).
Reconfiguration of the network by a modulatory interneuron may lead to
very substantial functional changes; for example, in the crustacean
stomatogastric system a new network is constructed from members of the
original pattern generating system (Meyrand et al.
1994
).
Role of OC interneurons in the feeding network
In previous models of the feeding system (Brierley et al.
1997a,b
; Elliott and Benjamin 1985a
,b
;
Yeoman et al. 1995
), production of the feeding pattern
only took place as long as the stimulation of the driving modulatory
interneuron (SO, N1L) was maintained. The longer-lasting (polycyclic)
excitatory effects of OC interneurons on protraction phase interneurons
(SO, N1L, N1M) means that the activity of the OC interneurons
contributes to the next cycle of feeding, therefore providing a
positive feedback from the swallow (N3) phase to the next feeding
cycle, which starts again with radula protraction (N1 phase).
The potential-dependent and long-lasting excitatory effect of OC
neurons may facilitate the initial excitatory trigger to the
protraction phase interneurons to evoke fictive feeding (Figs. 2 and
10). When the excitatory inputs from OC interneurons to N1 phase
interneurons are strong enough, they depolarize the membrane of the
follower up to the firing threshold, and finally the pattern-generating protraction phase interneurons evoke a feeding pattern. The electrical coupling between N1L and SO interneurons (Yeoman et al.
1995) further increases the excitatory effect of OC neurons on
the coupled partner.
After repetitive OC bursts, the excitatory component of the biphasic
response seen in the protraction phase interneurons (SO, N1L, and N1M)
facilitates. This means that repetitive stimulation of the OC
interneurons is more effective in stimulating these protraction phase
interneurons to drive fictive feeding. This is an additional feature of
the modulatory effect of the OC interneurons, specific to the
protraction phase (pattern-generating) interneurons. As this effect
lasts longer than the time for an individual feeding cycle, it may be
termed polycyclic modulation. This is a second example of polycyclic
excitatory modulation by the OC interneurons, adding to the ability of
the OC interneuron to enhance the synaptic output of the SO interneuron
(Elliott and Vehovszky 2000). In the Aplysia
feeding system an example of homosynaptic facilitation that modulates
the connections between an interneuron and its followers was described
recently (Sanchez and Kirk 2000
).
The most crucial question concerning the role of OC interneurons in feeding, however, is how these neurons are activated to produce their modulatory effects on the feeding system?
OC interneurons can be activated by the CPG system during feeding as they depolarize and contribute to the fictive feeding pattern after stimulation of protraction phase interneurons (Fig. 2, A-C). But this activation is unlikely to come from the known pattern-generating interneurons, as we have found inhibitory inputs from SO, N1L, and N1M interneurons to the OC interneurons (Fig. 11). The biphasic inputs (inhibition followed by depolarization) from N2v phase interneurons (Fig. 11E) or the excitatory electrical connection with other N3 phase (B4, N3p, N3t) feeding neurons (Figs. 4, 6, and 7) may also make some small contribution to the activation of the OC interneurons. However, it does not seem to be sufficient to trigger the OC burst.
OC interneurons also receive excitation from the CGCs, as do the B4,
N3p (and N3t) (Yeoman et al. 1996) swallowing phase
neurons, suggesting an additional extrinsic source of excitatory inputs from the cerebral ganglia. We cannot exclude, however, that the OC
interneurons are mainly activated from the periphery (through the
sensory system). Establishing this will require further studies in
semi-intact preparations, where the CNS is attached to peripheral organs (lip, mouth, buccal mass, salivary glands, etc).
OC interneurons are intrinsic modulators of the buccal feeding system
The OC interneurons cannot be considered as CPG interneurons
because a rhythm occurs even when they are silent. However, they definitely modulate the network. They receive rhythmic synaptic inputs
during fictive feeding and form connections with both CPG and
modulatory interneurons of the buccal ganglia but not with the cerebral
CGC. This means that the OC interneurons should be considered as
intrinsic rather than extrinsic modulators of the feeding network. Here
it is worth noting that the axonal branching of the OC, like that of
another intrinsic modulator, the SO interneuron, is extensive
throughout but confined to the buccal neuropil and does not enter other
parts of the CNS (Elekes et al. 1996; Elliott and
Benjamin 1985b
; Vehovszky et al. 1998
).
In the buccal ganglia of Lymnaea, known intrinsic modulators
as SO and N1L are cholinergic (Vehovszky and Elliott
1995; Yeoman et al. 1993
), while in the cerebral
ganglia, CBWC neurons contain APGWamide peptide (McCrohan and
Croll 1997
). The modulatory serotonin, however, arises from an
extrinsic modulator (CGC) neuron located in the cerebral ganglia
(Pentreath et al. 1982
), which can have polycyclic
effects on fictive feeding (Benjamin and Elliott 1989
). The OC interneurons are octopaminergic as their octopamine content and
octopaminergic synaptic contacts are already established
(Vehovszky et al. 1998
, 2000
).
In conclusion, through their synaptic connections with feeding motor neurons and interneurons, OC interneurons can modulate the activity and reconfigure the synaptic outputs of the buccal feeding system in Lymnaea. The effect produced by stimulating the OC interneurons is dependent on the activity (or quiescence) of the feeding network, due to the strongly biphasic outputs of the OC interneurons. As OC interneurons contain octopamine, this is the first example where an intrinsic member of the buccal feeding system in Lymnaea provides aminergic modulation.
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ACKNOWLEDGMENTS |
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This work was supported by Biotechnology and Biological Sciences Research Council Grant S08677.
Permanent address of Á. Vehovszky: Balaton Limnological Institute of the Hungarian Academy of Sciences, PO Box 35, Tihany H-8237, Hungary.
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FOOTNOTES |
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Address for reprint requests: C.J.H. Elliott, Dept. of Biology, University of York, PO Box 373, York YO10 5YW, UK (E-mail: cje2{at}york.ac.uk).
Received 31 August 2000; accepted in final form 1 May 2001.
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REFERENCES |
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