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INTRODUCTION |
In addition to generating
specific patterns of behaviorally relevant neural outputs, a major task
of the nervous system is to coordinate the functions of different
neural circuits so as to optimize behaviors. Neurons that project from
one region of the nervous system to distant regions provide unique
vantage points from which the coordination of neuronal function can be
studied. The projection neurons typically constitute a small subset of the total number of neurons within a region (Coleman et al.
1992
; Fredman and Jahan-Parwar 1979
;
Gillette et al. 1982
; Jing and Gillette
1999
; McCrohan and Kyriakides 1989
; Rosen
et al. 1991
). Such neurons may be important either in
coordinating different components of a single class of behavior or in
behavioral selection of unrelated or opposing behaviors. The responses
controlled by interganglionic neurons may be specific components of a
complex behavior or may be components of general responses that
represent the operation of a central arousal system. Previous findings
have indicated that feeding behavior in Aplysia involves
specific responses of the feeding apparatus (e.g., buccal mass) but
also involves considerable coordinated activity of the visceromotor
effectors that are controlled by the abdominal (Dieringer et al.
1978
; Koch et al. 1984
) and pedal-pleural
ganglia (Xin et al. 1996b
). We sought to identify
cerebral neurons that might be involved in coordinating aspects of
feeding behavior with behaviors regulated by the abdominal ganglia. We
used dye backfills of the abdominal connectives to define candidate
neurons. In the present research we report on the characteristics of
three identified neurons that appear to be involved in both buccal-mass
behaviors and visceral responses.
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METHODS |
Subjects
Experiments were done using wild-type Aplysia
californica weighing 200-300 g (Marinus, Long Beach, CA). A total
of approximately 200 animals were used. The animals were fed ad lib and
were maintained at 14-16°C in holding tanks containing aerated,
filtered artificial seawater (ASW) for at least 3-6 days before being
used for experiments. Two types of preparations were used in the
experiments: isolated ganglia, which included all the head ganglia
(buccal, cerebral, pedal, pleural) as well as the abdominal ganglion
and pleural-abdominal connectives, and semi-intact preparations that
included head ganglia and the head, including the lips, anterior
tentacle region, and cephalic artery, and buccal mass. In the
semi-intact preparations, the tentacle nerves and buccal nerves
remained intact. In another type of semi-intact preparation, the organs
associated with the innervation of the abdominal ganglion were retained
together with the circumesophageal and abdominal ganglia and the main
nerves of the abdominal ganglion (siphon, genital-pericardial,
branchial) and the pleural-abdominal connectives.
For dissection, animals were immobilized by injection of isotonic
magnesium chloride (25% of body weight). Isolated ganglia were pinned
to a clear silicone elastomer (Sylgard) floor of a recording chamber
containing fresh ASW. The cerebral and pedal-pleural ganglia were
pinned dorsal side up, and the abdominal ganglion was pinned dorsal or
ventral surface up, depending on the neurons to be investigated in a
particular experiment. In some experiments, the abdominal artery
remained attached to the abdominal ganglion via the pericardial nerve.
The semi-intact preparations were set in a clear acrylic plastic
(Lucite) recording chamber consisting of two compartments containing
ASW. The head ganglia were pinned in one compartment. The lips,
anterior tentacles, buccal mass, and cephalic artery that supplies the
lips and buccal mass were set in the second compartment. The second
compartment was deeper than the first so that the tissue could be
completely immersed in the ASW. The cephalic artery was cannulated, and
fresh ASW was pumped into the vascular system at a rate of about 0.5 ml/min to perfuse the tissue and to simulate the hydroskeleton of the animal. A suction tube for the outflow was set in the compartment to
control the fluid level. The isolated head could be presented with
mechanical or chemical stimuli. Mechanical stimuli were provided by the
tip of a heat-sealed glass Pasteur pipette that was applied manually to
a given receptive surface. Combined chemo-mechano stimuli consisted of
pieces of moistened dried-seaweed (laver, Vega Trading, New
York, NY) that were applied to the lips or tentacles with a fine, blunt
forceps. A pure chemical stimulus consisted of a seaweed extract
solution that was applied by a 1-ml syringe and slowly injected into
the ASW 1 cm from one side of the lip and tentacle region
(Susswein et al. 1978
). The partition between the two
compartments contained fine grooves that allowed the peripheral nerves
to pass through. The grooves were then filled with petroleum jelly
(Vaseline) to maintain a watertight seal between the two compartments.
Electrophysiology
All in vitro experiments were carried out at room temperature
(19-21°C). For the intracellular recording and stimulation, neurons
were impaled with double-barreled microelectrodes that were made of
thin-walled glass (World Precision Instruments, FL) and contained 2 M
potassium acetate. The electrodes were flow beveled so that their
impedance ranged from 10 to 15 M
. Neurons were identified based on
multiple criteria including size, morphology, coloration, position
within a ganglion, and synaptic interconnection to other cells. To test
for monosynapticity of connections, the ganglia were bathed in a high
divalent cation solution. Unless otherwise specified, this solution
contained 5× normal Ca2+ and 2× normal
Mg2+ [(in mM) 334 NaCl, 10 KCl, 50 CaCl2, and 100 MgCl2]. High divalent cations
raise the threshold for action potential generation although at the
concentrations that still permit intracellular firing of neurons, some
firing of interposed interneurons may occur (see, for example, Fig.
20B).
For extracellular recording or stimulation the cut ends of nerves were
drawn into small-diameter polyethylene suction electrodes. Nerve
recordings were made with an AC amplifier (A-M Systems), and electrical
stimulation of the nerves was provided by a Grass S88 stimulator. For
monitoring contractions of blood vessels, an arm of an isotonic tension
transducer (Harvard Bioscience) was attached to the distal end of the
vessel (Xin et al. 1996a
).
Morphology
To determine the sizes, shapes, and destinations of axons of
identified neurons, cells were filled with dye. For these experiments, the potassium acetate in the stimulating electrode was replaced by a
solution of 3% 5(6)-carboxyfluorescein dye (Kodak) in 0.1 M potassium
citrate, titrated to pH 8.0 with KOH (Rao et al. 1986
). The electrodes were beveled so that the impedance of the electrode containing the dye was 15-20 M
, and the impedance of the potassium acetate electrode was 10-15 M
. Successful intracellular labeling was achieved by iontophoretic injection of the dye for 15-60 min, followed by a 48-h incubation at 4°C to allow the dye to fully fill
the processes. To reduce active transport of the dye from the cells
during incubation (Rosen et al. 1991
; Steinberg
et al. 1987
), the bathing ASW solution included 10 mM (final
concentration) probenecid (Sigma). The living ganglia were cleared in
50% glycerol in ASW. Fluorescence was visualized with a Nikon
fluorescence microscope. Backfills of the pleural-abdominal connectives
were done using biocytin (Sigma), as previously described
(Xin et al. 1999
).
Immunocytology for serotonin combined with intracellular dye
labeling
To label identified cells with dye, 5% Lucifer yellow was
injected into cells by hyperpolarizing current. The ganglia were fixed
in 4% paraformaldehyde, 0.1 M phosphate buffer (PBS; pH 7.4), 30%
sucrose at room temperature for 2 h, then washed overnight with
0.1 M PBS, 30% sucrose at 4°C. The immunocytology was carried out
using a modification of previously described techniques (Lloyd et al. 1985
; Longley and Longley 1986
). The
fixed and washed ganglia were preincubated at room temperature for
2 h with 1% normal goat serum (NGS; Miles Science, Naperville,
IL) in 0.1 M PBS to reduce nonspecific binding of primary antibody. The
ganglia were then incubated in rabbit serotonin antiserum (Sigma)
diluted 1/500 in PBS containing 1% NGS for 2 days at 4°C; washed in
PBS at 4°C for 1 day, and incubated at 4°C for 1 day in goat
anti-rabbit IgG rhodamine-conjugated Fab fragment diluted 1/50 (Cappel,
Malvern, PA). Finally, the ganglia were washed in PBS again at 4°C
for 1 day, then mounted on slides and coverslipped with Aqua-Poly Mount
(Polyscience, Warrington, PA). The ganglia were viewed under a Nikon
microscope equipped for epifluorescence and were photographed with
Tri-X film. In one series of immunocytological studies, a rat
antiserotonin antibody obtained from F. S. Vilim (Mt. Sinai School
of Medicine) was used and the results were identical to those obtained
with the rabbit antibody.
Controls for specificity of staining
Six ganglia were used for controls. Replacement of primary or
secondary antibody did not result in staining. Dopaminergic cells that
are revealed by formaldehyde-glutaraldehyde-induced fluorescence
(Goldstein and Schwartz 1989
) were not observed in the C
cluster following fixation in paraformaldehyde.
 |
RESULTS |
There are three CAIs in the C cluster of the cerebral ganglion
Backfills of the pleural-abdominal connectives revealed up to 50 cerebral neurons that send an axon into one or both the connectives. The number of cells filled was highly variable, but there appeared to
be at least three relatively large neurons (50-100 µm) that typically were seen in the medial region of the cerebral C cluster. We
designate these cells as CC neurons (CC2, CC3, and CC7) to refer to the
fact that they are in the cerebral ganglion C cluster. The C cluster
(Jahan-Parwar and Fredman 1976
) consists of a
compact group of cells (see Fig. 2) located in the caudal region of the dorsal surface of the cerebral ganglion, just rostral and slightly medial to the group of large neurons referred to as the B cluster (Jahan-Parwar and Fredman 1976
; Teyke et al.
1989
). As explained later, the cell we refer to as CC3 appears
to be the same cell that was previously reported to be located in the
cerebral B cluster (Hawkins 1989
; Mackey et al.
1989
). CC2 and CC3 are bilateral cells, whereas CC7 has only
been found unilaterally in the right hemiganglion.
CC2 is a monopolar neuron that sends its axon to the ipsilateral
cerebral-pleural connective. A main branch continues down into the
abdominal ganglion via the pleural-abdominal connective. Another branch
goes to the ipsilateral pedal ganglion and then travels to the
contralateral pedal ganglion via the pedal-pedal commissure (Fig.
1A). CC3 is a bipolar neuron
that sends one axon to the ipsilateral cerebral-pleural connective and
the other to the contralateral cerebral-pleural connective (Fig.
1B). In the pleural ganglia, the main axons continue
bilaterally to the abdominal ganglion. The axon sends branches to the
pedal ganglia. CC7 is located in the right cerebral C cluster. The cell
is presumed to be unilateral since it has never been encountered in the
left hemiganglion despite extensive searches. CC7 sends its axon across to the contralateral pleural ganglion via the contralateral
cerebral-pleural connective (Fig. 1C). In the pleural
ganglion, the axon divides and sends one branch to the abdominal
ganglion via the pleural-abdominal connective. The other branch travels
to the ipsilateral pedal ganglion and continues to the contralateral
pedal-pleural ganglion via the pedal-pedal commissure.

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Fig. 1.
Schematic drawings illustrating the position and axon distribution of 3 cerebral-abdominal interneurons located in the cerebral C cluster. The
drawings are based on visualizations of neurons after fills with
5(6)-carboxyfluorescein dye. A: example of CC2 and its
processes (n = 6). A symmetrical cell is present in
each hemi-ganglion. CC2 sends its axon down to the ipsilateral pleural
ganglion. The axon bifurcates and one branch enters the
pleural-abdominal connective; the other branch enters the pleural-pedal
commissure, continues through the ipsilateral pedal ganglion and then
continues into the pedal-pedal commissure. The axon branch appears to
terminate in the contralateral pedal ganglion. B:
example of CC3(CB-1) and its processes (n = 8). A
symmetrical cell is present in each hemi-ganglion. Shortly after
leaving the cell body, the axon of CC3 bifurcates (see Fig.
2A). One axon enters the ipsilateral cerebral-pleural
connective; the other axon enters the contralateral cerebral-pleural
connective. In the pleural ganglion, each axon bifurcates and sends 1 branch to the pedal ganglion, and the other branch into the
pleural-abdominal connective. C: example of CC7 and its
processes (n = 8). CC7 has only been encountered in
the right cerebral hemiganglion and is presumed to be a unilateral
neuron. The axon of CC7 crosses to the contralateral cerebral ganglion
and continues down the contralateral cerebral-pleural connective into
the pleural ganglion and then, via the pleural-pedal commissure and the
pedal-pedal commissure it continues into the ipsilateral and
contralateral pedal ganglia. A branch of the axon then continues into
the contralateral pleural ganglion where it appears to terminate. ATn,
anterior tentacular nerve; C-BC, cerebral buccal commissure; C-PC,
cerebral-pedal commissure; C-PlC, cerebral-pleural commissure; LLABn,
lower labial nerve; P, pedal nerve; Pl-AbC, pleural-abdominal
connective; P-P comm., pedal-pedal commissure; ULABn, upper labial
nerve.
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Neuron CC3 is immuno-positive for serotonin
The morphology of CC3 was studied by means of carboxyfluorescein
or Lucifer yellow dye injection Fig.
2A shows Lucifer yellow labeling of CC3, which is shown at its typical location within the C
cluster. The label clearly shows CC3 has a short initial segment which
bifurcates into two axons. At higher magnifications, the axons could be
traced into the ipsilateral and contralateral cerebral-pleural and
pleural-abdominal connectives.

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Fig. 2.
CC3 is immunopositive for serotonin. The figure shows a dorsal view of
the right anterior and middle region of the cerebral ganglion. CC3 was
intracellularly labeled with Lucifer yellow and the preparation was
then stained for serotonin. A: visualization with a
Lucifer yellow filter set revealed CC3 ( ) and its characteristic
bifurcating axon. B: visualization with a rhodamine
filter set revealed cells that are immunopositive for serotonin. The
large immunopositive cell in the anterior region of the ganglion
(top) is likely the metacerebral cell (MCC). A
small number of cells, up to five, in each C cluster were
immunopositive. In this example, 2 serotonin-positive cells were
stained in each C cluster. One of the cells in the right cluster ( )
coincides with the Lucifer-filled CC3 neuron. Low-level background
staining reveals the other neurons that comprise the right C cluster
(for orientation within the ganglion see the position of CC3 indicated
in Fig. 1B). The left border of
B shows 2 immunopositive cells located in the left
cerebral C cluster. Based on its position, the lower, stained cell in
the left hemiganglion is likely to be the left homologue of CC3
(n = 5, calibration bar: 100 µm).
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Intracellular injection of CC3, CC2, or CC7 with Lucifer yellow,
followed by immunostaining for serotonin, revealed that CC3 was the
only cerebral-abdominal interneuron in the C cluster that was
immunopositive for serotonin. Figure 2B shows an example in which CC3 was first identified by its characteristic morphology, position, and synaptic connections. The cell was then labeled with
Lucifer yellow and subsequently shown to be stained with a serotonin
antibody (Fig. 2B). As many as five cells immunopositive for
serotonin (5-HT) were stained in the C cluster (cf.
Wright et al. 1995
), but neither CC2 nor CC7 was
immunopositive. CC3 is the largest, most posterior cell of the five
serotongeric cells in the C cluster. The 5-HT staining pattern (up to 5 immunopositive cells) was similar to those observed in two other
gastropod mollusks, Pleurobranchaea and Tritonia
(Sudlow et al. 1998
). In one experiment, we labeled both
the right and left CC3 with Lucifer yellow and found that they both
subsequently stained for serotonin.
In a previous study, it was reported that there is a serotonergic
cerebral-abdominal interneuron that is located in the B cluster of the
cerebral ganglion and consequently was named CB-1 (Hawkins
1989
; Mackey et al. 1989
). Since CC3 appeared to
have the same distinctive axonal morphology as CB-1 (Mackey et
al. 1989
; Wright et al. 1995
), we sought to
determine if there might be two similar serotonergic cells, one located
in the C cluster and a second in the B cluster. The B cluster is
composed of relatively large neurons spaced relatively far apart,
whereas the C cluster is a compact group of small and medium sized
cells located just anterior to the B cluster cells. Provided that there
is no significant damage to the cells during desheathing, the C cluster
forms a clearly demarcated distinct group. To determine if there is a serotonergic B cluster cell that might have properties similar to CC3,
we stained for serotonin in a series of eight ganglia. We found,
however, that in none of these ganglia were any serotonin positive
cells found in the B cluster. In all cases, serotonergic neurons that
were located on the dorsal surface of the posterior region of
the cerebral ganglion were found to be restricted to the C cluster.
Consequently we conclude that CC3 is likely to be identical to the
previously described CB-1 neuron. We suggest (see
DISCUSSION) that the full designation of CC3 be CC3(CB-1).
All three CAI cells in the cerebral C cluster get inputs from
stimuli applied to the head and lips
Feeding behavior in Aplysia can be evoked by tactile
and/or chemical stimuli applied to the anterior regions of the animal (Kupfermann 1974
). We found that stimulation of the lips
or anterior tentacles with a piece of moist seaweed or a glass rod
evoked excitatory synaptic input into CC2 (Fig.
3, A and B). There
was no significant difference between touching the lip or tentacle with
a piece of moist seaweed compared with a polished glass probe. The
responses evoked by inputs to CC2 occurred with a relatively short
latency (less than 100 ms), well before any sign of a rhythmic buccal
ganglion responses, which typically have latencies of 3-10 s. CC3 also
receives excitatory input when the lip is stimulated (Fig.
4, A and B),
whereas CC7 is inhibited (Fig.
5A). CC2 and CC3 were similar
not only with regard to their responses to stimuli applied to the head,
but they also received similar input during evoked buccal-mass
movements that were initiated by applying brief tactile stimuli to the
buccal mass. During evoked cycles of forward and backward movement,
both CC2 and CC3 became tonically active (Figs. 3C and
4C). Electrical stimulation of buccal nerve 2, which can
elicit rhythmic buccal nerve activity or buccal mass movements (Nargeot et al. 1997
), also produced tonic activity in
CC2 and CC3 during the time that rhythmic buccal activity occurred as judged by buccal nerve recordings (Figs. 3D and
4D). In contrast to CC2 and CC3, rhythmic buccal activity
was associated with a different pattern of input to CC7. CC7 showed
phasic activity. Spiking in CC7 was absent during the phase of each
retraction movement of the buccal mass or radula (Fig. 5B).
We also determined if CC7 fired phasically during a nonfeeding rhythmic
program, namely a locomotor program. To examine the firing of CC7
during rhythmic locomotor programs, fictive locomotion was elicited by electrical stimulation of pedal nerve 9 (P9). Fictive locomotion was
monitored by extracellular recording from P11 (also called the pedal
artery shortener nerve). This nerve contains the axon of the pedal
artery shortener neuron, which fires in phase with locomotor programs
(Skelton and Koester 1992
; Xin et al.
1996b
). In contrast to the strong phasic activity seen during
buccal mass movements, CC7 showed tonic firing following the
stimulation of P9, and it continued to fire during the fictive
locomotor program (Fig. 5C). The firing of CC7 showed some
variation, but this was not clearly phase locked to the locomotor
rhythm recorded in P11.

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Fig. 3.
CC2 receives sensory inputs from the lips and buccal ganglion.
A: in a simplified preparation consisting of the lips
and the buccal mass together with the cerebral and buccal ganglia,
touching the lips with a moistened piece of seaweed ( ) produced
excitation in CC2 (n = 4). B: when
CC2 was hyperpolarized, touching the lips evoked numerous fast
excitatory postsynaptic potentials (EPSPs, n = 4).
C: touching the buccal mass ( ) evoked buccal mass
movements and an increase in the tonic firing of CC2
(n = 3). D: electrical stimulation
of buccal nerve 2 (BN2) evoked a buccal motor program as evidenced by
rhythmic activity recorded with an extracellular electrode on buccal
nerve 2. CC2 showed a burst of activity immediately following the
shock, and then showed irregular spiking not distinctly in phase with
the BN2 activity (n = 3).
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Fig. 4.
CC3 receives inputs from the lips and buccal ganglion.
A: touching the lips with a piece of moist seaweed
evoked excitation in CC3 (n = 4). B:
when CC3 was hyperpolarized, touching the lip evoked numerous fast
EPSPs (n = 4). C: during buccal mass
forward and backward movements evoked by direct touch of the buccal
mass, CC3 fired tonically (n = 3).
D: electrical stimulation of buccal nerve 2 (BN2) evoked
rhythmic activity in buccal nerve 2 and irregular firing of CC3 not
distinctly in phase with BN2 activity (n = 4).
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Fig. 5.
CC7 receives inhibitory inputs from the lips and buccal ganglion.
A: touching the lips with a piece of moist seaweed ( )
evoked brief inhibition of CC7 spiking (n = 4).
B: in preparations of head ganglia with the buccal mass
attached, during buccal mass forward and backward movements, CC7
exhibited activity that was in phase with buccal mass movements. CC7
spiking strongly decreased during the retraction phase of the radula
and buccal mass movement (observed visually and marked with horizontal
lines; n = 3). C: electrical
stimulation of pedal nerve 9, which produces a locomotor program,
increased CC7 firing rate. P11, which contains the axon of the pedal
artery shortener neuron, reflects the rhythmic activity of a fictive
locomotor program. The firing of CC7 was not distinctively in phase
with the fictive locomotor program (n = 3).
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Table 1 summarizes the basic
morphological and input features of the cerebral-abdominal interneurons
in the cerebral C cluster.
Firing of cerebral C cluster CAI cells influences cells that appear
to be involved in buccal mass activity
To explore the possible involvement of the C-cluster CAIs in
feeding, we fired individual cells and recorded possible spike activity
of units that project to the buccal ganglion via the cerebral-buccal
(c-b) connectives. Firing of the CAIs evoked activity of several cells
that were recorded extracellularly in the ipsilateral and contralateral
c-b connectives (Figs.
6-8).
The evoked activity may be mediated by interneurons in the
pleural-pedal or abdominal ganglia since it was eliminated by cutting
the cerebral-pleural and -pedal connectives. Additional evidence for a
possible role of the CAIs in feeding was the finding that CC7 inhibited
C-PR and in turn was inhibited by firing of cerebral-pedal
regulator neuron (C-PR) (Fig. 9). C-PR is
involved in generating aspects of appetitive feeding responses such as
the head-up posture and lengthening of the neck (Teyke et al.
1990
).

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Fig. 6.
Firing of CC2 evokes neural activity in the cerebral-buccal connectives
(n = 4). In this example, the left CC2 (LCC2) was
fired.
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Fig. 7.
Firing of CC3 evokes neural activity in the cerebral-buccal connectives
(n = 4). In this example, the right CC3 (RCC2) was
fired.
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Fig. 8.
Firing of CC7 evokes neural activity in the cerebral-buccal connectives
(n = 3). Since CC7 is located in the right
hemiganglion, the RCBC represents the ipsilateral connective.
R, right; L, left; CBC, cerebral-buccal connective.
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Fig. 9.
CC7 and cerebral-pedal regulator neuron (C-PR) mutually inhibit one
another. A: effect of firing CC7 on C-PR.
B: effect of firing C-PR on CC7. Discrete synaptic
potentials one for one with CC7 or C-PR spikes were not observed in
these experiments (n = 3).
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Effects of CC2 on left upper quadrant cells and other neurons in
the abdominal ganglion
Firing of CC2 was found to excite or inhibit numerous cells in the
abdominal ganglion. Some of the cells that are excited by CC2
apparently send axons into the genital nerve since brief firing of CC2
evoked a prominent burst of unit activity that was recorded from the
genital nerve (Fig. 10). Among cells
that send their axons into the genital nerve, we found that CC2
produced one-for-one excitatory postsynaptic potentials (EPSPs, Fig.
11A) in the left upper
quadrant cells (LUQs), which appear to be motor neurons for the renal
pore and cause it to close (Koester and Alevizos 1989
).
The EPSPs persisted in a high divalent cation solution (Fig.
11B), suggesting that CC2 is monosynaptically connected to
LUQ cells.

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Fig. 10.
Firing of CC2 evokes a burst of neural activity recorded in the genital
nerve of the abdominal ganglion (n = 4).
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Fig. 11.
CC2 monosynaptically excites abdominal ganglion left upper quadrant
(LUQ) cells. A: firing of CC2 produced 1-for-1 EPSPs in
an LUQ cell. The LUQ cell was slightly hyperpolarized to prevent
spontaneous firing and enhance the size of the EPSP. Note that because
of the long distance between the cerebral and abdominal ganglia, the
EPSPs had a long latency. B: the EPSPs persisted in a
high divalent cation solution (3× Mg2+, 3×
Ca2+; n = 6).
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CC2 strongly excited the L9 gill motor neurons (Fig.
12). The excitation was associated with
the presence of EPSPs that were not one-for-one with the CC2 spikes and
outlasted the firing of CC2. Thus the excitation that CC2 produced was
likely due to the activity of one or more interneurons. Since CC2
appeared to be the only C cluster cerebral-abdominal interneuron that
strongly excited L9 cells, the presence of excitatory input to L9 could be used as one of the identifying features of CC2.

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Fig. 12.
CC2 has strong excitatory effects on gill motor neuron L9 cells. Firing
of CC2 strongly excited both L91 and L92
(n = 5). The excitation was associated with the
occurrence of fast EPSPs that were not 1-for-1 with CC2 spikes and
outlasted the firing of CC2, indicating that the excitation is very
likely polysynaptic (n = 3). Because CC2 appears to
be the only neuron in the C cluster of the cerebral ganglion found to
strongly excite L9 cells, L9 was often used to identify CC2.
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Since L9 is excited as part of a defensive reflex (Kupfermann
and Kandel 1969
), we recorded from L9 together with cerebral Bn
(B cluster, narrow spike) cells, which also appear to be involved in
defensive responses (Teyke et al. 1989
). We found that
while L9 was excited by CC2, Bn cells exhibited a polysynaptic
inhibition (Fig. 13A).
Similarly, simultaneous recording from L9 and the gill motor neuron
LDG revealed that when L9 was excited, LDG was inhibited (Fig.
13B). Thus both the excitation of L9 and the inhibition of LDG and Bn outlast CC2's firing for the same duration, consistent with
the possibility that there is an interposed interneuron presynaptic to
all of these cells.

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Fig. 13.
CC2 inhibits cells involved in withdrawal responses. A:
firing of CC2 inhibits Bn cells, cerebral ganglion neurons that are
involved in head withdrawal responses (n = 3).
B: CC2 inhibits LDG cells, which are abdominal ganglion
neurons that are involved in gill withdrawal. In these experiments an
L9 cell was also recorded and showed its characteristic excitation in
response to firing of CC2 (n = 4).
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Firing of CC2 appeared to modestly increase the rate of firing of the
"spontaneous" EPSP (Fig.
14A) that can be recorded in bursting neuron R15 (Koester et al. 1993
;
Kupfermann 1970
). The EPSPs were not one for one with
the CC2 spikes, suggesting that the effect was polysynaptic. It was
previously shown that one source of rhythmic EPSPs to R15 is
interneuron XIII, which is located in the right pedal ganglion
(Koester et al. 1993
). To test whether the excitatory
effect of CC2 on R15 might be mediated by interneuron XIII, we cut the
right pleural-abdominal connective that is the pathway by which
interneuron XIII extends its axon to the abdominal ganglion. When the
connective was cut, as expected, spontaneous EPSPs in R15 were no
longer present. Nevertheless firing of CC2 still excited R15 even in
the absence of any prominent fast EPSPs (Fig. 14B). Another
possible neuron that could mediate an excitatory effect of CC2 onto R15
is L40, an abdominal ganglion interneuron that excites interneuron XIII
as well as L9 cells (Koester et al. 1993
). We found that
firing of CC2 excited L40 (Fig.
15A). As revealed by
hyperpolarization of L40, the excitation evoked by CC2 did not appear
to be monosynaptic but was associated with a burst of EPSPs that were
not one for one with CC2 spikes (Fig. 15B). The excitatory
effect on the presumptive interneuron that excites L40 was not
invariably observed. For example in one series of runs, CC2 affected
L40 in only approximately 50% of the trials.

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Fig. 14.
Firing of CC2 excites the abdominal ganglion R15 neuron.
A: R15 usually exhibits regular rhythmic bursting in the
isolated abdominal ganglion; firing of CC2 increased the firing rate of
a spontaneously firing excitatory interneuron that is often seen in
recordings from R15 (n = 3). One type of EPSP seen
in R15 is known to be produced by activity of interneuron XIII, which
is a unilateral cell, located in the right pedal ganglion and sends its
axon to the abdominal ganglion only via the right pleural-abdominal
connective. B: cutting the right connective eliminated
the EPSP in R15 neuron as seen in the figure, but firing of CC2 still
excited R15, suggesting that the effect of CC2 on R15 may not only
involve interneuron XIII (n = 3).
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Fig. 15.
The effect of CC2 on L9 may involve L40, an abdominal ganglion
interneuron that excites L9. A: firing of CC2 produced a
strong repeatable excitatory input onto L40. B: when L40
was strongly hyperpolarized, it was revealed that firing of CC2
increased the rate of a fast EPSP in L40, suggesting that CC2 excites
an interneuron which in turn excites L40 (n = 2).
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Effects of CC3 on L10, LUQ cells and other abdominal ganglion cells
Firing of CC3 was found to produce effects on neurons that project
out to various nerves including pedal-ganglion nerves such as pedal
nerve 3 and abdominal ganglion nerves, most prominently the genital
nerve (Fig. 16). Examination of
identified neurons in the abdominal ganglion revealed that one neuron
that receives prominent excitation when CC3 is fired, is L11 (Fig.
17), a cell that sends a large axon
into the genital nerve (Koester and Kandel 1977
). The
EPSP evoked in L11 has a slow onset and decay. Fast trace recordings
showed it was not associated with any obvious fast potentials (Fig.
17D). When CC3 was fired in bursts every 2-3 s, the evoked
slow EPSP in L11 was little changed from burst to burst (Fig.
17A). CC3 evoked a similar slow excitation of abdominal ganglion interneuron L10 (Fig. 17, B and C).

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Fig. 16.
Firing of CC3 evokes activity in peripheral nerves of the abdominal and
pedal ganglia. This figure shows a simultaneous recording from CC3 with
pedal ganglion nerve 3 and abdominal ganglion genital nerve
(n = 4).
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Fig. 17.
CC3 excites identified neurons in the abdominal ganglion.
A: excitatory effect of firing of CC3 on L11.
B: excitatory effect of firing of CC3 on L10.
C and D: the recordings at faster sweep
speeds; the excitatory input evoked in L10 and L11 is not associated
with any visible fast EPSPs, suggesting that the cells are excited by a
slow potential (n = 6).
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L10 is known to monosynaptically inhibit LUQ cells (L2-L5)
(Frazier et al. 1967
) in which it evokes a
characteristic dual-phase (fast/slow) inhibitory postsynaptic potential
(IPSP). We found that firing of CC3 evoked a similar IPSP in LUQ cells
(Fig. 18). Typically after a variable
delay of approximately 100-400 ms, small discrete IPSPs, not
one-for-one with CC3 spikes, were observed together with a large, slow
hyperpolarization.

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Fig. 18.
CC3 inhibits abdominal ganglion LUQ cells. As shown in the figure, in
some instances CC3 produced fast IPSPs without a prominent slow phase.
In other LUQ cells, CC3 evoked IPSPs that exhibited a prominent
summating slow component in the absence of fast components (see Fig.
21A; n = 5). We have not attempted
to analyze the source of these different effects, which could be
characteristic of the specific LUQ cell (Evans et al.
1991 ) or could be related to some experiment-specific property
of the LUQ cell, such as its membrane potential.
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CC3 is present bilaterally in the cerebral ganglion. Because the left
and right abdominal hemiganglia are highly asymmetrical, we examined
whether there were any major differences between the ipsilateral and
contralateral connections of CC3. For these experiments, both the left
and right CC3 cells were impaled in the same ganglion, and their
effects on selected abdominal ganglion cells were examined. We found
that both the left and right CC3 inhibited LUQ cells (Fig.
19A), and both the left and
right CC3 cells excited L10 (Fig. 19B).

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Fig. 19.
CC3 neurons have effects on abdominal ganglion cells ipsilateral and
contralateral to the CC3 cell. A: LUQ cells receive
inhibitory input from both the ipsilateral and contralateral CC3 cells.
In this example, the LUQ cell exhibited the rhythmic bursting that is
often seen in LUQ cells. B: L10 cells receive excitatory
input from both the ipsilateral and contralateral CC3
(n = 3).
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To test for the possible monosynapticity of the actions of CC3 on L10,
L11, and LUQ cells, we examined it actions in a high divalent cation
solution. In this solution, the slow excitation to L10 persisted (Fig.
20, A and B), but
the excitation to L11 and inhibition of LUQ cells did not persist (data
not shown). These results are consistent with the conclusion that the
CC3 connection to L10 may be monosynaptic, whereas the effects of CC3
on the LUQ cells are polysynaptic, arising, at least in part, from the
firing of L10.

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Fig. 20.
The synaptic connection of CC3 to L10 persists in the presence of a
high divalent cation solution. A: in normal artificial
seawater (ASW), firing of CC3 excited L10 strongly. B:
when the abdominal ganglion was bathed in a high divalent cation
solution (3× normal Mg2+ and 3× normal Ca2+),
firing of CC3 still depolarized the membrane potential of the L10
neuron, suggesting that CC3 produces a slow monosynaptic excitatory
input to L10. The decreased firing of L10 in the high divalent cation
solution, compared with ASW, may be a consequence of an increased
threshold for spiking in the presence of the high divalent cations
(n = 3).
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Another abdominal ganglion neuron that receives synaptic input from L10
is R15 (Kandel et al. 1967
; Koester and Kandel
1977
), and as expected, R15 was excited by firing of CC3. When
R15 was in its burst mode, firing of CC3 produced a shortening of the interburst interval (Fig.
21A). In fast sweep speed
recordings in which the R15 neuron was slightly hyperpolarized to
suppress spontaneous spiking, firing of CC3 resulted in spiking of R15 and increased the frequency of a spontaneous EPSP in the cell (Fig.
21B).

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Fig. 21.
CC3 produced weak excitation of R15. A: firing of CC3
decreased the interburst interval of R15. An LUQ cell was
simultaneously recorded to aid in identifying CC3. B:
when R15 was hyperpolarized, firing of CC3 revealed an increase in the
firing of an excitatory interneuron (n = 5).
C: when interneuron XIII (which fires rhythmically and
directly excites R15) was hyperpolarized, firing of CC3 still increased
the firing rate of R15 even in the absence of any visible fast EPSPs
(n = 2).
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In addition to excitatory input from L10, R15 is excited by interneuron
XIII, a cell located in the right pedal ganglion (Koester et al.
1993
). When interneuron XIII and R15 neurons were recorded from
simultaneously, we found that although firing of CC3 excited R15, it
had no effect on interneuron XIII (Fig. 21C).
Another neuron excited by L10 is the heart motor neuron
RBHE (Mayeri et al. 1974
). As
expected, firing of CC3 resulted in excitatory input to
RBHE (Fig.
22A). In addition, we found
that CC3 may weakly excite an R20 cell (Fig. 22A) and LBvc
(Fig. 22B); and it inhibited L9 (Fig. 22B). None
of these effects were associated with one-for-one synaptic potentials.

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Fig. 22.
Firing of CC3 had synaptic effects on a diverse set of neurons in the
abdominal ganglion. A: CC3 excited the heart exciter
cell, RBHE, and produced weak excitation of a cell
identified as R20. B: CC3 evoked excitatory input to an
LBvc cell, while it inhibited an L9 cell (n = 3).
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Effects of CC7 on LBvc cells and other abdominal cells
In a semi-intact preparation that included the organs innervated
by the abdominal ganglion, firing of CC7 was observed to evoke a large
contraction of the abdominal artery (Fig.
23). The contraction appeared to
involve the circular muscles of the artery since the diameter of the
artery was reduced markedly and there was a virtual shut-down of the
flow of fluid that was perfused through the artery. Because the
abdominal artery is innervated by the LBvc motor neurons in the
abdominal ganglion (Mayeri et al. 1974
), we tested for a
connection between CC7 and the LBvc cells. We found that CC7 excites
all three LBvc cells. Figure 24 shows
an example of the EPSP that CC7 produced in a LBvc cell. The EPSPs
appeared to be monosynaptic. They were one-for-one with the CC7 spikes
and had a constant latency. Furthermore the EPSPs persisted in a high
divalent cation solution (Fig. 24B).

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Fig. 23.
Firing of CC7 evoked strong contraction of the abdominal artery. CC7
was intracellularly stimulated at 15 Hz. The contraction of the
abdominal artery was monitored by an isotonic displacement transducer
(n = 6).
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Fig. 24.
CC7 excites LBvc cells, which are motor neurons for the abdominal
artery. A: firing of CC7 produced 1-for-1 EPSPs in an
LBvc cell in normal seawater. B: the EPSPs persisted in
a high divalent cation solution (n = 5).
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In addition to evoking contractions of the abdominal artery, firing of
CC7 resulted in contraction of the heart, and we found that CC7
produced one for one EPSPs in the heart motor neuron, RBHE (Fig.
25A). The EPSPs in the
RBHE cell persisted in a high divalent cation
solution (Fig. 25B), suggesting that CC7 monosynaptically excites this neuron.

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Fig. 25.
CC7 excited RBHE, a heart excitatory neuron.
A: in normal seawater, firing of CC7 produced 1-for-1
EPSPs in RBHE. B: the EPSPs persisted in a
high divalent cation solution (n = 3).
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Although CC7 appears to exert its main actions on elements of the
cardiovascular system, it also has effects on other abdominal neurons
that do not appear to be directly involved in cardiovascular function.
It produced a weak slow excitation of LUQ cells (Fig. 26). It also evoked a multiphasic
synaptic input (excitatory-inhibitory-excitatory) to R20. In addition
to affecting abdominal ganglion neurons it has diverse actions on
numerous neurons located in the pedal (Fig. 27, A and
B) and pleural ganglia (Fig.
28, A and B).

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Fig. 26.
Firing of CC7 excited LUQ cells and produced signs of a weak complex
input to neuron R20 (n = 3). The traces from the 3 cells were obtained in different experiments, and a train of typical
frequency and duration of CC7 spikes is shown in the bottom
trace.
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Fig. 27.
Firing of CC7 excites or inhibits pedal ganglion neurons.
A: example of a pedal neuron (Pn1) that was excited by
firing of CC7 (n = 7). B: example of
a pedal neuron (Pn2) that was inhibited by firing of CC7
(n = 7).
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Fig. 28.
The observed effects of CC7 on pleural neurons were all excitatory.
A: some actions of CC7 were associated with a slow EPSP
in pleural neurons. B: some unidentified pleural
ganglion cells exhibited a short-latency excitation, and
hyperpolarization of these pleural neurons revealed that CC7 could
evoke short-latency fast EPSPs (n = 6).
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The synaptic outputs of the cerebral-abdominal interneurons (CAIs) to
different functional systems and identified cells in the abdominal
ganglion are summarized in Table 2.
 |
DISCUSSION |
The overall data (Tables 1 and 2) suggest that although each of
the CAIs in the cerebral C cluster has distinctive properties, they
also have certain shared features. Thus the CAIs may each have
individual distinctive behavioral functions but may also be active
conjointly in certain similar behaviors.
The properties of CC2 and CC3 suggest that they are more closely
related to each other than to CC7. All three CAIs receive synaptic
inputs when the lips and tentacles are contacted with relatively gentle
tactile stimulation such as that provided by sea weed. The input,
however is excitatory for CC2 and CC3 but is inhibitory for CC7. The
firing of the three CAIs produces activation, albeit weak, of neurons
that send an axon into the cerebral-buccal connective. It is likely
that the activated cells are cerebral-buccal interneurons (CBIs), and
it remains to be determined if the same set of cells are affected by
each of the three CAIs.
The CAIs all alter the activity of abdominal ganglion neurons, but
examination of the specific neurons affected indicates that each CAI
influences a distinctive group of cells. Each of the three cells has a
prime site of action, involving monosynaptic connections (indicated in
bold in Table 2) and in addition affects overlapping groups of cells by
means of polysynaptic connections.
CC2 and CC3 may affect kidney function
CC2 has monosynaptic excitatory connections to the LUQ cells,
whereas CC3 has a monosynaptic connection to interneuron L10 (Frazier et al. 1967
) and indirectly inhibits LUQ cells,
at least in part by means of the inhibitory connections that L10 makes onto LUQ cells (Kandel et al. 1967
). CC2 and CC3 may be
an example of an "antipodal" pair of interneurons, that is, pairs
of interneurons that exert actions that are largely opposite in sign,
as is the case for example of the CC6/CC5 pair of cerebral neurons
(Xin et al. 1996b
, 2000
). The group of LUQ cells and L10
also exert opposing actions on the renal pore. LUQ cells promote
closing of the kidney pore and L10 promotes opening of the kidney pore (Koester and Alevizos 1989
). Thus firing of CC2 would be
expected to promote closing of the kidney pore, whereas firing of CC3
should promote opening of the kidney pore. CC2 and CC3 also have
opposing polysynaptic actions on gill motor neurons. CC2 evokes
excitatory inputs into L9 cells, while CC3 evokes inhibitory inputs to
L9 cells.
In these experiments, we did not specifically attempt to examine the
significance of serotonin as a possible neurotransmitter of CC3. In
Aplysia it is now clear that serotonin is involved in a
variety of functions, including roles in both aversive (Clark and Kandel 1993
; Sun and Schacher 1998
) as well
as appetitive behaviors (Lloyd et al. 1984
). Given the
suggestion that CC3 may be involved in some aspects of feeding
behavior, it would be interesting to explore the role of serotonin on
neurons that may be involved in ingestive aspects of feeding or perhaps
in defensive behaviors that might compete with feeding (Jing and
Gillette 2000
).
In previous work, a serotonergic cell termed CB-1 was suggested to be
involved in heterosynaptic facilitation of the EPSPs of siphon sensory
neurons in the abdominal ganglion and to be involved in dishabituation
and sensitization of gill and siphon withdrawal responses
(Hawkins 1989
; Mackey et al. 1989
;
Wright et al. 1995
). Because in our studies, CC3 was
always found in the C cluster and never in the B cluster, we assumed
that this was not CB-1 and was therefore a previously unidentified
neuron. Our attempts to locate a serotonergic neuron in the B cluster, however, were unsuccessful, and based on its overall physiological and
morphological characteristics, we have concluded that CC3 is very
likely identical to CB-1. In fact, CC3 is located in the posterior
region of the C cluster, adjacent to the anterior region of the B
cluster and might have been visualized as part of the B cluster.
Because the prefix "CB" implies CB-1 is in the B cluster, to avoid
possible confusion about the location of the cell, we suggest that the
full designation for the cell be CC3(CB-1).
Because CC3(CB-1) may be involved in defensive responses elicited by
noxious stimuli, it is possible that its actions on cerebral-buccal interneurons reflects a role in egestive feeding responses that occur
when the animal encounters noxious or inedible food stimuli. It is not
possible, however, to exclude multiple roles for this cell, such as a
general arousal function as suggested for serotonergic neurons in
Pleurobranchaea (Jing and Gillette 2000
). For
example, it might be involved in aspects of appetitive arousal in
response to relatively mild stimuli applied to the lips but might be
involved in defensive arousal in response to strong or noxious stimuli. The putative cerebral-buccal interneurons that are excited by the CAIs
have not been identified so that it is not known what the likely buccal
program or programs are associated with activity of the CAIs. Indeed
cerebral-buccal interneurons appear to be involved in both ingestive
(Perrins and Weiss 1998
; Rosen et al. 1991
) as well as egestive (Rosen et al. 1991
,
1998
) buccal motor programs.
The fact that the serotonergic neuron CC3 in turn excites another
serotonergic neuron RBHE as well as neurons
involved in diverse behaviors is reminiscent of an organization of a
serotonergic arousal network that may be present in
Pleurobranchaea (Jing and Gillette 2000
) and
Clione (Satterlie and Norekian 1996
). In
gastropod mollusks, a limited number of serotonergic cerebral neurons
such as As4 in Pleurobranchaea excite other serotonergic
neurons and may provide a form of general arousal (Jing and
Gillette 2000
). CC3 in Aplysia may be analogous or
perhaps even homologous to cells such as As4 of
Pleurobranchaea (Jing and Gillette 1999
), dorsal swim interneurons of Tritonia (Sudlow et al.
1998
), cerebral ganglion Cd3 cell in Lymnaea
(Croll and Chiasson 1989
), and CPA1, CPB1 in
Clione (Panchin et al. 1995
).
CC7 affects the vasoconstrictor/heart system
The overall pattern of inputs and outputs of CC7 suggests that it
plays a functional role quite different from that of CC3 and CC2. CC7
has a strong monosynaptic excitatory connection to vasoconstrictor
neurons, and its firing can evoke a substantial constriction of the
abdominal aorta. Furthermore CC7 excites the serotonergic heart motor
neuron RBHE. CC3 can also excite
RBHE, but its actions are polysynaptic, mediated
most likely by its excitation of L10. CC7, by contrast, appears to
monosynaptically excite RBHE. An important
functional difference between CC7 and CC2/CC3 is the nature of the
synaptic inputs they receive. Tactile stimuli to the head evoke
inhibition in CC7 instead of excitation as seen in CC2 and CC3.
Furthermore the synaptic inputs CC7 receives in association with buccal
programs are strongly phasic, whereas the inputs to CC2 and CC3 are
tonic and not distinctly correlated with the phase of the buccal
program. Since the firing of CC7 produces a substantial constriction of
the abdominal aorta, CC7 has the properties appropriate for a cell that
can mediate or at least contribute to the previously described
correlation between rhythmic variations of blood flow and movements of
the buccal mass that occur during feeding (Koch et al.
1984
). It has been suggested that this gating of blood flow
during buccal mass movements may aid in the generation of functional
biting movements (Koch and Koester 1982
; Koch et
al. 1984
). Thus CC7 may provide a means by which a somatic
neural control system is coordinated with visceral responses that
provide support for the somatic behaviors [see also data on neuron
CLE1 in Clione (Arshavsky et al. 1992
)]. In this sense, CC7 may function to optimize feeding movements and therefore can be considered an important component of the neural basis
of food-induced arousal. It is significant that although CC7 fires in a
strongly rhythmic mode during a rhythmic feeding motor program, it
fires in a tonic mode during a rhythmic locomotor program. Thus CC7 may
provide another example of a cell such as the previously described
cells CC5 and CC6 (Xin et al. 1996b
, 2000
), that are
active in multiple behaviors, but play different roles in the various behaviors.
CC7 may be identical to a previously described neuron in
Aplysia termed CB2 (Wright et al. 1995
). CB2
is located near CC3(CB-1). Like CC7, CB2 is not serotonergic and sends
its axon into the contralateral pleural-abdominal connective. It is not
known whether CB2 is present only unilaterally, and because its
synaptic connections have not been described, it is not possible to
definitively identify it as identical to CC7.
This work was supported by National Institutes of Health Grants
MH-50235, 36730 MH-12890, DA-07135, GM-320099, and K05-MH-01427 and by
National Science Foundation Grant IBN 9808411.
Address for reprint requests: I. Kupfermann, New York State Psychiatric
Institute, 1051 Riverside Dr., Box 87, New York, NY 10032 (E-mail:
ik7{at}columbia.edu).