Effect of a Serotonergic Extrinsic Modulatory Neuron (MCC) on
Radula Mechanoafferent Function in Aplysia
VERA ALEXEEVA, 1 DMITRY BOROVIKOV, 1 MARK W. MILLER, 3 STEVEN C. ROSEN, 4 AND
ELIZABETH C. CROPPER 1,2
1
Department of Physiology and Biophysics and 2The Fishberg Center for Research in Neurobiology, The Mt. Sinai
Medical Center, New York, New York 10029; 3 Institute of Neurobiology and Department of Anatomy, University of
Puerto Rico Medical Sciences Campus, San Juan, Puerto Rico 00901; and 4Center for Neurobiology and Behavior,
The New York State Psychiatric Institute, New York, New York 10032
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
INTRODUCTION
A striking feature of behavior is its plasticity. Even relatively simple motor acts are often continuously adjusted to
accommodate changes in the external environment. To characterize cellular mechanisms that are likely to mediate this
type of plasticity, interactions between sensory neurons and
central pattern generators (CPGs) have been studied both
in vertebrate and invertebrate preparations (e.g., Burrows
1997; Buschges 1995; Clarac and Cattaert 1996; Laurent
1991; Pearson and Ramirez 1997; Sillar 1991; Wallen
1997). From this work, it has become apparent that sensory
input can play an important role in determining the characteristics of physiologically relevant motor programs. An important aspect of sensory control of rhythmic movement,
however, is that in many systems sensory-induced changes
in rhythmic activity must occur in a manner that is appropriate for the ongoing behavior. In these cases therefore
afferent-CPG interactions are often not just unidirectional
transfers of information. Instead, as afferent activity can influence CPG activity, CPG activity can often in turn regulate
afferent activity (e.g., El Manira et al. 1991a; Sillar and
Skorupski 1986; Skorupski and Sillar 1986; Vinay et al.
1996; Wolf and Burrows 1995). Although progress has been
made in characterizing mechanisms by which CPG activity
can modify afferent transmission, the range of these mechanisms appears to be extensive (e.g., Sillar 1991) and is just
beginning to be appreciated (Pearson and Ramirez 1997).
In recent work we have begun to address this issue within
the context of feeding in the marine mollusk Aplysia. One
potential source of modulation of sensory neurons are the
serotonergic metacerebral cells (MCCs). The MCCs (or homologous neurons) have been well characterized in mollusks
(e.g., Berry and Pentreath 1976; Cottrell and Macon 1974;
Gerschenfeld and Paupardin-Tritsch 1974; Gerschenfeld et
al. 1978; Weinreich et al. 1973) and have been extensively
studied within the context of feeding (e.g., Gillette and Davis
1977; Granzow and Kater 1977; McCrohan and Audesirk
1987; Weiss and Kupfermann 1976; Weiss et al. 1982; Yeoman et al. 1994a,b, 1996). These cells exert widespread
actions that are manifested both centrally and peripherally,
i.e., on the feeding circuitry and on feeding musculature.
0022-3077/98 $5.00 Copyright q 1998 The American Physiological Society
/ 9k2d$$oc20 J203-8
09-15-98 19:29:19
neupas
LP-Neurophys
1609
Downloaded from http://jn.physiology.org/ by 10.220.32.246 on June 17, 2017
Alexeeva, Vera, Dmitry Borovikov, Mark W. Miller, Steven C.
Rosen, and Elizabeth C. Cropper. Effect of a serotonergic extrinsic
modulatory neuron (MCC) on radula mechanoafferent function in
Aplysia. J. Neurophysiol. 80: 1609–1622, 1998. The serotonergic
metacerebral cells (MCCs) and homologous neurons in related mollusks have been extensively investigated within the context of feeding.
Although previous work has indicated that the MCCs exert widespread actions, MCC modulation of sensory neurons has not been
identified. We characterized interactions between the MCCs and a
cell that is part of a recently described group of buccal radula mechanoafferents. The cell, B21, has a peripheral process in the tissue
underlying the chitinous radula [the subradula tissue (SRT)]. Previous studies have shown that B21 can fire phasically during ingestive
motor programs and provide excitatory drive to the circuitry active
during radula closing/retraction. We now show that activity of B21
can be modulated by serotonin (5-HT) and the MCCs. Centrally,
although a slow depolarization is typically recorded in B21 as a result
of MCC stimulation, this depolarization does not cause B21 to spike.
It can, however, increase B21 excitability enabling a pulse that was
previously subthreshold to elicit an action potential in B21. B21 is in
fact rhythmically depolarized during the radula closing/retraction
phase of ingestive motor programs. Thus central effects of the MCCs
on radula mechanoafferent activity are only likely to be apparent
while B21 is receiving input from the feeding central pattern generator.
Peripherally, radula mechanoafferent neurons can be activated 1)
when a mechanical stimulus is applied to the biting surface of the
SRT and 2) when the SRT contracts. MCC stimulation and 5-HT
modulate B21 responses to both types of stimuli. For example, MCC
stimulation and low concentrations of 5-HT cause subthreshold mechanical stimuli applied to the SRT to become suprathreshold. 5HT and MCC stimulation also enhance SRT contractility. Peripheral
effects of MCC activity are also likely to be phase dependent. For
example, MCC stimulation does not cause B21 to respond to peripheral stimuli with an afterdischarge. Consequently, radula mechanoafferents are likely to be activated when food is present between the
radula halves during radula closing/retraction but are not likely to
continue to fire as opening/protraction is initiated. In a similar vein,
MCC effects on the contractility of the SRT will only be apparent
when contractions are elicited by motor neuron activity. SRT motor
neurons are rhythmically activated during ingestive motor programs.
Thus we have shown that radula mechanoafferent activity can be
modulated by the MCCs and that this modulation is likely to occur
in a phase-dependent manner.
1610
ALEXEEVA, BOROVIKOV, MILLER, ROSEN, AND CROPPER
/ 9k2d$$oc20 J203-8
This depolarization will therefore potentiate afferent transmission during this phase of behavior.
In investigating the effects of MCC activity on B21 we
therefore sought to determine whether MCC activity was
likely to affect the central mechanism that gates radula mechanoafferent activity. Thus we sought to determine whether
the MCCs would directly depolarize B21 and cause it to
spike and/or whether the MCCs would simply increase the
excitability of B21. Additionally, radula mechanoafferent
activity is influenced by contractions of the subradula tissue
(SRT) (Cropper et al. 1996b), which contains the processes
of B21 (Miller et al. 1994). The SRT is innervated by at
least one motor neuron that fires rhythmically during ingestive feeding motor programs (Borovikov et al. 1997).
When the SRT contracts, neuron B21 is activated, and its
responsiveness to touch is increased (Cropper et al. 1996b).
Thus in this study we additionally sought to determine
whether contractile properties of the SRT were altered by
MCC activity and whether changes in the contractile properties of the SRT were likely to affect radula mechanoafferent
activity. Some of these data were published as an abstract
(Cropper et al. 1996a).
METHODS
Animals
Experiments were conducted with 250–350 g A. californica that
were maintained in holding tanks containing 14–167C artificial
seawater (ASW). Animals were anesthetized by injection of isotonic MgCl2 and then dissected to create reduced preparations.
Identification of neurons
The nomenclature used in this study follows that of Gardner
(1971). The radula mechanoafferent cluster of neurons was identified
by criteria described by Miller et al. 1994. It consists of a centrally
located group of cells on the rostral surface of the buccal ganglion.
Radula mechanoafferent neurons are electrically coupled to each
other, and peripherally generated spikes are elicited when the radula
is touched. Neurons B21 and B22 are the largest cells of the cluster
(Rosen et al. 1992). Neuron B21 differs from neuron B22 in that 1)
it is generally larger, 2) it is typically more spherical, neuron B22 is
typically more elongate, 3) it is generally more ventrally located, and
4) IPSPs are recorded from neuron B21 when the B4/B5 neurons
are stimulated. In contrast, neuron B22 receives excitatory synaptic
input from neurons B4/B5 (Rosen et al. 1992).
Serotonergic input to the SRT and the buccal ganglion
Localization of 5-HT-immunoreactive ( 5-HT-IR ) fibers was
determined by standard whole-mount methods ( Longley and
Longley 1986; Miller et al. 1994 ) . The primary antiserum
( rabbit host, Incstar, Stillwater MN ) was applied for 48 h
( 1:200 dilution, room temperature ) and the second F ( ab * ) 2
tetramethyl rhodamine isothiocyanate – conjugated antibody
( anti-rabbit IgG heavy and light chain, donkey host; Jackson
Immunoresearch, West Grove, PA ) was applied for 24 h ( 1:200
dilution, room temperature ) . Tissues were viewed with a Nikon
microscope equipped with epifluorescence and photographed
with Tri-X ( ASA 400 ) film. Within the buccal ganglion, B21
and B22 were identified by using the criteria listed above. They
were injected with 3% Lucifer yellow ( LY ) dye ( 20 – 30 min,
1 s on, 1 s off pulses ) before fixation ( 4% paraformaldehyde,
09-15-98 19:29:19
neupas
LP-Neurophys
Downloaded from http://jn.physiology.org/ by 10.220.32.246 on June 17, 2017
Peripheral effects of serotonin (5-HT) or MCC activity have
been extensively investigated in Aplysia in a few model
neuromuscular systems (e.g., Fox and Lloyd 1997; Lotshaw
and Lloyd 1990; Scott et al. 1997; Weiss et al. 1978). Although central effects of the homologous CGCs have recently begun to be investigated in a related mollusk, Lymnaea (Yeoman et al. 1996), central effects of MCC activity
in Aplysia have not been well characterized (although see
Weiss et al. 1978). Sensory neurons with central or peripheral somata that are modulated by MCC activity have not
been identified.
The sensory neuron in this study is the SCP-containing
radula mechanoafferent B21 (Miller et al. 1994; Rosen et
al. 1992). This neuron is of interest because B21 and other
radula mechanoafferents appear to play an important role in
ingestive feeding behavior in Aplysia (Cropper et al. 1996b;
Miller et al. 1994; Rosen et al. 1992–1994). For example,
it was suggested that these neurons are partially responsible
for producing the adjustments in feeding motor programs
that are necessary to convert bites (where food is not ingested) to bite–swallows (where food is deposited in the
esophagus) (Cropper et al. 1996b; Rosen et al. 1992). This
conversion is accomplished by changes in phase and amplitude relationships between radula closing/retraction and radula opening/protraction. For example, radula closing/retraction is prolonged and enhanced, and radula opening/protraction is delayed to accommodate the enhanced closing/
retraction (Cropper et al. 1990). That B21 could play a role
in inducing these types of changes in ingestive responses is
suggested by the fact that it is activated when the biting
surface of the radula is touched, e.g., by food (Miller et al.
1994; Rosen et al. 1992). Additionally, it is either directly
or indirectly connected to many of the identified motor neurons and interneurons active during ingestive motor programs (Rosen et al. 1992–1994). Many neurons active during closing/retraction are directly excited by radula mechanoafferent activity, and many neurons active during opening/
protraction are indirectly inhibited by radula mechanoafferent activity.
Although B21 clearly functions as a sensory neuron it is
additionally rhythmically depolarized by the feeding CPG
(Miller et al. 1994; Rosen et al. 1993, 1994). It is likely
that these CPG-induced depolarizations in B21 play an important role in gating the effectiveness of centripetal activity
in this cell (Rosen et al. 1993, 1994). The mechanism utilized in this context is, however, not the mechanism commonly observed in situations where CPGs induce primary
afferent depolarizations (PADs). Thus PADs often exert a
local inhibitory effect because they are often mediated by a
presynaptic conductance increase that decreases the effectiveness of afferent transmission to postsynaptic neurons
(e.g., Clarac and Cattaert 1996; Nusbaum et al. 1997). In
contrast, CPG-induced depolarizations in B21 are not strictly
presynaptic, i.e., they can be recorded in the soma of B21,
and they exert a potentiating effect on afferent transmission
(Rosen et al. 1993, 1994). Thus when B21 is depolarized
centripetal spikes are more, rather than less, effective at
eliciting PSPs in follower cells. During ingestive motor programs B21 is rhythmically depolarized during radula closing/retraction (Miller et al. 1994; Rosen et al. 1993, 1994).
SEROTONERGIC MODULATION OF MECHANOAFFERENT ACTIVITY
1611
4 h, room temperature ) and processed for 5-HT immunocytochemistry. Ganglia were examined on a scanning confocal microscope ( NORAN Odyssey ) equipped with an argon laser and
barrier filters suitable for viewing the LY ( band pass 520 – 560
nm) and rhodamine ( long pass 590 nm) . Images were generated
with the Metamorph Imaging System ( West Chester, PA ) .
They were transferred as TIFF bitmap files for processing with
Adobe Photoshop and labeled with CorelDraw.
To determine whether serotonergic input to the SRT did indeed
arise from the MCCs, electrophysiological techniques were used
to determine whether intracellular MCC stimulation elicited extracellular spikes in peripheral sections of the radular nerve. Intracellular stimulation techniques used were standard. Electrodes were
beveled to relatively low impedances (i.e., õ10 MV ) and were
filled with 2 M potassium acetate. Extracellular recordings were
made with suction electrodes.
Effects of MCC stimulation and 5-HT on B21 afferent
activity and contractile properties of the SRT
These experiments were conducted in reduced preparations
that were described in detail elsewhere ( e.g., Cropper et al.
1996b ) ( see also Fig. 4A ) . Briefly, the SRT was removed from
the chitinous radula, and other muscles, nerves, and ganglia were
/ 9k2d$$oc20 J203-8
dissected away. Particular dissections depended on the purpose
of the experiment. For example, the cerebral ganglion was only
present in experiments where effects of the MCCs were studied.
In experiments where SRT contractions were elicited by motor
neuron stimulation, both the radular nerve and buccal nerve 3
were intact. In experiments where SRT contractions were elicited by stretch, only the radular nerve was intact. In all cases,
preparations were transferred to Sylgard-lined dishes, and the
SRT was attached to a semiisotonic force transducer ( Harvard
Apparatus ) . A Lucite subchamber was then placed around the
SRT to pharmacologically isolate it from the nervous system
( Fig. 4A ) . Contractions of the SRT were elicited either by
stretch or with intracellular stimulation of B66 ( the SRT motor
neuron ) . Stretch was induced by counterweighting muscles.
Thus, the SRT was not directly attached to the transducer ( Fig.
4 A ) . It was attached to one end of a lever. The middle of this
lever made contact with the transducer and was therefore immobilized and served as a pivot point. Counterweights were applied
by placing metal washers on the end of the lever that was not
attached to the SRT. In experiments where afferent activity of
B21 was studied, the SRT was not attached to the force transducer. It was pinned to a Sylgard ‘‘stage’’ so that it was approximately perpendicular to the device used to apply mechanical
stimuli. Mechanical stimuli were delivered by means of a mini-
09-15-98 19:29:19
neupas
LP-Neurophys
Downloaded from http://jn.physiology.org/ by 10.220.32.246 on June 17, 2017
FIG . 1. The metacerebral cell (MCC) is a source of serotonergic input to the SRT. A: intracellular stimulation of the
MCC (bottom traces) elicits extracellular responses (top traces) in distal sections of the radular nerve. B: serotonin (5HT) immunoreactivity in the radular nerve secondary branch that innervates the subradula tissue (SRT). A large smooth
immunoreactive fiber (arrow) is present in the nerve. Small varicose fibers appear to be associated with the nerve sheath.
Calibration bar 100 mm. C: 6–8 fine immunoreactive fibers pass in parallel (arrow) in the distal radular nerve branch within
the SRT. Near the terminus of this branch, the serotonergic fibers bear off in various directions and subdivide, reaching a
substantial portion of the tissue. Calibration bar 100 mm. D: lower magnification shows the high-density of serotonergic
innervation in the region of the SRT adjacent to the insertion of the accessory radula closer (ARC) muscle. The density of
immunoreactive fibers tapers off within the ARC muscle itself. Calibration bar 250 mm.
1612
ALEXEEVA, BOROVIKOV, MILLER, ROSEN, AND CROPPER
speaker ( Quam) that had a wooden probe ( tip diameter 1 mm)
that was perpendicularly attached to the speaker membrane. Reproducible movements of the membrane were regularly elicited
by driving the speaker with a Grass stimulator.
A second type of preparation was used in experiments where
contractions of the SRT were elicited with extracellular nerve
stimulation. The SRT was left attached to the chitinous radula,
except that, to gain access to the SRT, the I2 muscle and the
radula sac were removed. Additionally, connections between the
SRT and muscles I4, I5, and I7 were severed to reduce the
likelihood that movements not specifically related to the contraction of the SRT would be transduced. The radular nerve was
generally left intact; all other buccal nerves were cut close to
the buccal ganglion so that stimulating electrodes could easily
be applied. Transducers were attached to the SRT and chitinous
radula with a piece of thread ( as described previously ) . Buccal
/ 9k2d$$oc20 J203-8
nerves were stimulated with suction electrodes and a Grass stimulator.
cAmp measurements in the SRT
Animals were anesthetized and dissected, and the SRT and
attached musculature were removed from the chitinous radula. To
specifically isolate the part of the SRT that contains the peripheral
processes of B21, we removed as much of the I4 and I5 muscles
as possible. The SRT was trimmed and left for 2 h in ASW to
allow for recovery from adenosine 3 *,5 *-cyclic monophosphate
( cAMP) increases induced by the dissection. Exogenous 5-HT was
applied for 10 min. The SRT was then homogenized in 65% ethanol, boiled for 15 min, and spun at 10,000 g for 15 min. cAMP
levels were measured with a commercially available radioimmuno-
09-15-98 19:29:19
neupas
LP-Neurophys
Downloaded from http://jn.physiology.org/ by 10.220.32.246 on June 17, 2017
FIG . 2. 5-HT-IR fibers in the buccal ganglion. A : low-power confocal image of the rostral surface of the buccal
ganglion. Ganglion is oriented with the buccal commissure directed toward the top right corner of the image. Neurons
B21 and B22 in the radula mechanoafferent cluster were identified by using morphological and electrophysiological
criteria and injected with Lucifer yellow ( LY, arrows ) before processing the ganglion for 5-HT immunoreactivity
( TRITC-conjugated second antibody ) . In this image, the barrier filter used to view LY allowed ‘‘bleedthrough’’ of the
rhodamine-labeled 5-HT-IR fiber system. B : same field as A using a filter that permits viewing the rhodamine-labeled
5-HT fibers exclusively. Positions of the cell bodies of B21 and B22 are indicated as in A . Neurons with asterisks are
likely to be B4 / B5. Calibration bar of 100 mm applies to A and B . C : higher magnification of the midline region of
ganglion using the same filter as A . B21 and B22 are spindle-shaped cells with prominent processes directed medially
and laterally. Note that after the immunocytochemistry process LY retention within the nuclei ( n ) of injected cells
exceeds that of the cytoplasmic regions, which exhibit a ‘‘halo’’ appearance. Arrows indicate the large initial segments
of B21 and B22 that are directed toward the midline. Calibration bar 50 mm. D : higher-power image of the region
shown as a dotted box in C . Varicose 5-HT-IR fibers ( arrows ) are associated with the initial segment and cell body of
B21. Calibration bar 20 mm.
SEROTONERGIC MODULATION OF MECHANOAFFERENT ACTIVITY
1613
The MCCs branch extensively within the buccal ganglion,
so it was of interest to determine whether they have processes
that terminate on B21 centrally, i.e., within the buccal ganglion. To determine the relation between 5-HT-IR fibers and
radula mechanoafferent neurons, B21 and B22 neurons were
injected with LY dye, and buccal ganglia were processed
for 5-HT immunocytochemistry (Fig. 2). 5-HT-IR fibers
were observed in the vicinity of B21/B22, particularly in
the region of the medially directed initial segment of B21
(Fig. 2D). In the following physiological experiments we
therefore sought to determine whether the MCCs act centrally and/or peripherally to modulate radula mechanoafferent activity.
Central effects of MCC stimulation
assay (RIA; Amersham). cAMP levels are expressed as picomoles
per milligram of tissue (wet weight).
RESULTS
Source of serotonergic input to the SRT
Previous studies have suggested that the serotonergic
MCCs innervate the SRT. At least in proximal sections of
the radular nerve, extracellular spikes can be recorded when
the MCCs are stimulated intracellularly (Weiss and Kupfermann 1976). The radular nerve does, however, branch several times before it reaches the SRT. We therefore placed
extracellular electrodes as close to the SRT as possible and
stimulated the MCCs. Extracellular responses were recorded
in peripheral branches of the radular nerve (Fig. 1A).
Additionally, we used immunocytochemical techniques to
determine whether there are 5-HT-IR fibers in regions of the
SRT that contain the receptive fields of radula mechanoafferent neurons. 5-HT-IR fibers were most obvious in the region
of the SRT where the accessory radula closer muscle inserts
(Fig. 1D). This is also a region that is innervated by B21
(Rosen et al. 1992). Thus the current physiological and
anatomic observations suggest that the MCCs are likely to
be the source of serotonergic input to the SRT.
/ 9k2d$$oc20 J203-8
Effects of 5-HT on contractile properties of the SRT
One method of eliciting contractions of the SRT is by
stretch ( Cropper et al. 1996b ) . To determine whether 5HT affects stretch-induced contractions of the SRT, we
used preparations in which the SRT was separated from
the chitinous radula and the rest of the buccal mass. The
SRT was placed in a subchamber and attached to a semiisotonic force transducer ( Fig. 4 ) . The transducer arm
was adjusted so that the SRT was counterweighted with
loads that elicited small infrequent contractions. 5-HT was
then applied directly to the SRT subchamber. Stretch-induced contractions increased in size and frequency at low,
presumably physiological, concentrations ( i.e., 10 08 –
10 06 , n Å 3; Figs. 4 and 5 ) . These effects were seen
09-15-98 19:29:19
neupas
LP-Neurophys
Downloaded from http://jn.physiology.org/ by 10.220.32.246 on June 17, 2017
FIG . 3. Central effects of MCC stimulation. A: depolarizations of a few
millivolts were observed in neuron B21 when the MCC was stimulated at
relatively high firing frequencies. Because neuron B21 is generally ¢10
mV from threshold, MCC stimulation did not directly elicit action potentials
in B21. These depolarizations could be observed when experiments were
conducted in a 31 Mg 2/ ; 31 Ca 2/ artificial seawater (ASW). B: when
B21 was repeatedly depolarized with subthreshold fixed current pulses, the
pulses evoked spikes when the MCC was stimulated. These effects on
excitability could be observed when the MCCs were fired at frequencies
that did not produce central depolarizations.
To determine whether MCC input is likely to directly
cause B21 to spike we stimulated MCCs and recorded the
resulting change in membrane potential in B21 (Fig. 3).
Firing the MCC at relatively high frequencies resulted in a
slow depolarizing response of a few millivolts in B21 in five
of six preparations (e.g., Figs. 3A and Fig. 6). B21 is generally ¢10 mV from threshold in quiescent preparations (e.g.,
Fig. 3B), and the depolarization evoked by the MCC failed
to evoke spikes. It is difficult to unequivocally determine
whether the effect of the MCCs on B21 is direct because
neurons that are electrically coupled to B21 such as B15
and B16 are also depolarized by MCC stimulation (Weiss
et al. 1978). Effects of MCC stimulation on B21 could,
however, still be observed when experiments were conducted in a 31 Mg 2/ ; 31 Ca 2/ ASW (Fig. 3A).
We next sought to determine whether MCC stimulation
could produce an increase in the excitability in neuron B21,
such that it exhibited enhanced firing during an input that
occurs during feeding motor programs. Depolarizing current
pulses were repeatedly injected into B21 before, during, and
after MCC stimulation (Fig. 3B). Current pulses that were
subthreshold before MCC stimulation elicited action potentials in neuron B21 during MCC stimulation and just after
stimulation in three of four preparations. These data suggest
therefore that, although the MCCs are not likely to directly
cause radula mechanoafferent neurons to fire, MCC activity
may alter B21 excitability and thereby alter its response
during feeding motor programs.
1614
ALEXEEVA, BOROVIKOV, MILLER, ROSEN, AND CROPPER
when activity in the central nervous system was blocked
by incubating the nervous system in 0.51 Ca 2/ ; 41 Mg 2/
solutions ( e.g., Fig. 4C2 ) . In general the effects of 5-HT
were long lasting, and it generally took an hour
for preparations to recover from higher concentrations
of 5-HT.
Although it is not known if the counterweight used in
these experiments simulated a physiological load, the radula
undergoes vigorous movements and shape changes during
feeding in intact animals (Drushel et al. 1997), and the SRT
is likely to experience considerable loads. We found that the
effects of 5-HT could be seen even with very light counterweights (e.g., 45 mg), suggesting that 5-HT could induce
contractions of the SRT in the absence of activity of the
SRT motor neuron. To determine whether 5-HT is released
from the MCCs in concentrations that are sufficient to exert
this type of effect on the SRT, the SRT was attached to the
force transducer with a counterweight that was just barely
subthreshold for eliciting contractions, and the nervous system was placed in a 41 Mg 2/ ; 0.51 Ca 2/ ASW. Under these
conditions MCC stimulation caused the SRT to contract, but
the contractions were much smaller than those elicited by
/ 9k2d$$oc20 J203-8
direct application of 5-HT to the SRT (n Å 5; Fig. 6).
These data suggest that MCC-induced contractions are not
therefore likely to produce vigorous radula movements.
In addition to stretch, SRT contractions can be elicited by
firing of a SRT motor neuron, B66 (Borovikov et al. 1997).
We therefore next determined whether 5-HT could affect
SRT contractions evoked by stimulation of buccal nerve 3,
which contains the axon of the SRT motor neuron. Experiments were conducted in relatively intact buccal mass preparations. 5-HT applied directly to the SRT increased nerve
3-evoked contraction size in a concentration-dependent manner (n Å 3; Fig. 7). We next determined whether MCC
activity could modulate parameters of muscle contractions
evoked by direct firing of B66. Contractions of the SRT
were therefore elicited with a fixed number of spikes before,
during, and after MCC stimulation (n Å 4; Fig. 8A). Contraction size, rate of rise, and relaxation rate all increased.
The MCCs do therefore modulate parameters of motor neuron-elicited contractions of the SRT.
To determine whether MCC-induced changes in the
contractile properties of the SRT could alter radula mechanoafferent activity, contractions of the SRT were elicited
09-15-98 19:29:19
neupas
LP-Neurophys
Downloaded from http://jn.physiology.org/ by 10.220.32.246 on June 17, 2017
FIG . 4. 5-HT affects contractions of the
SRT elicited by stretch. A: reduced preparation used to study effects of stretch on
the SRT. In these preparations the SRT was
separated from the chitinous radula, placed
in a subchamber pharmacologically isolated from the nervous system, and attached
to a semiisotonic force transducer. The tissue was stretched by adjusting the position
of a counterweight. B: stretch-induced contractions of the SRT produce centripetal
spikes in the left and right B21 neurons
(LB21 and RB21). C: 5-HT increases the
frequency and amplitude of stretch-elicited
contractions. In C1 the ganglion was bathed
in normal ASW. The counterweight was
adjusted to produce small rhythmic contractions, and 10 07 M 5-HT was applied to
the SRT subchamber. Stretch-induced contractions increased in amplitude and frequency. C2: to determine whether the effect shown in C1 was mediated centrally
or peripherally, the experiment shown in
C1 was repeated with the ganglion bathed
in 0.51 Ca 2/ ; 41 Mg 2/ .
SEROTONERGIC MODULATION OF MECHANOAFFERENT ACTIVITY
1615
FIG . 5. Effects of 5-HT on stretch-induced contractions of the SRT are
concentration dependent. The preparation used for this experiment was
similar to the one shown in Fig. 4A except that the nervous system was
not present. All 4 records are from the same preparation. The preparation
was washed for 30 min between 5-HT applications (not shown). 5-HT
was applied directly to the SRT subchamber. At low concentrations 5-HT
increased the amplitude and frequency of stretch-induced contractions of
the SRT. At 10 05 M, however, contractions were inhibited.
with different parameters of motor neuron stimulation,
and changes in B21 responsiveness were determined ( Fig.
8 B ) . When the intraburst stimulation frequency was increased contractions were altered in the most MCC-like
manner, i.e., contractions produced with higher intraburst
stimulation frequencies were larger in amplitude and had
a faster rate of contraction. Under these conditions the
number of centripetal spikes in neuron B21 did in fact
increase. Taken together therefore the data shown in Fig.
8, A and B , suggest that effects of the MCCs on contractile
properties of the SRT are in fact likely to alter radula
mechanoafferent activity.
of Aplysia (e.g., Lloyd et al. 1984; Lotshaw and Lloyd 1990;
Weiss et al. 1979). To test this hypothesis exogenous 5-HT
was applied to the SRT for 10 min, and resulting cAMP
levels were measured. 5-HT did indeed increase SRT cAMP
levels in a concentration-dependent manner (Fig. 9A). Additionally we tested a membrane permeable cAMP analogue (8
CPT–cAMP) on physiological preparations. 8 CPT–cAMP
exerted 5-HT–like effects on parameters of contractions of
the SRT (Fig. 9C). Exogenous 5-HT was still effective at
elevating cAMP levels in a Ca-free ASW (Fig. 9B), i.e.,
Mechanism of action of 5-HT on the SRT
The relatively slow onset and long-lasting nature of the
effects of 5-HT on contractile properties of the SRT suggested that 5-HT might be acting via a second messenger.
Specifically, we hypothesized that effects might be cAMP
mediated because it has been commonly demonstrated that
5-HT elevates cAMP in other muscles of the buccal mass
/ 9k2d$$oc20 J203-8
FIG . 7. 5-HT modulates contractions of the SRT elicited by nerve shock.
Contractions of the SRT were repeatedly elicited by shocking buccal nerve
3 (bars under recordings). 5-HT increased contraction amplitude at 1007
and 10 06 M.
09-15-98 19:29:19
neupas
LP-Neurophys
Downloaded from http://jn.physiology.org/ by 10.220.32.246 on June 17, 2017
FIG . 6. Effects of MCC stimulation on a SRT subjected to stretch. The
preparation used for these experiments is shown in Fig. 4A. The SRT
was counterweighted in a manner that was just subthreshold for eliciting
contractions, and the MCC was stimulated. Contractions that were much
smaller than those elicited by exogenous 5-HT were elicited (arrows). This
was the case even when the MCCs were fired in different patterns, e.g., in
repeated bursts. Because MCC stimulation produces excitability increases
centrally (e.g., note the depolarization in B21), it was possible that elicited
contractions resulted from the activation of a SRT motor neuron. The experiment was therefore repeated with the ganglion, but not the SRT, bathed in
41 Mg 2/ ; 0.51 Ca 2/ . Small contractions were still elicited with MCC
stimulation.
1616
ALEXEEVA, BOROVIKOV, MILLER, ROSEN, AND CROPPER
under conditions where transmitter release presumably cannot occur, suggesting that the 5-HT effect was direct.
Effects of 5-HT and the MCCs on the peripheral
excitability of B21
One type of experiment that suggests that 5-HT can act
outside the buccal ganglion and affect the peripheral excitability of B21 is shown in Fig. 10. Here 5-HT was sequentially applied to the SRT subchamber at different concentrations while B21 responses were recorded. At 10 08 M SRT
contractions were elicited, and centripetal spikes were recorded in neuron B21. At 10 07 M, contractions were similar
in size, but fewer centripetal spikes were recorded in neuron
B21. Finally at 10 06 M large SRT contractions still occurred,
/ 9k2d$$oc20 J203-8
but centripetal spikes were no longer recorded from neuron
B21. As the 5-HT is washed out, however, B21 responses
returned (Fig. 10B). Although in this experiment it cannot
be determined whether 5-HT exerted an excitatory action
on the responsiveness of B21 at lower concentrations, an
inhibitory effect was observed at 10 06 M.
To determine whether 5-HT can affect responsiveness of
B21 to peripherally applied mechanical stimuli, a stimulus that
was barely suprathreshold was repeatedly applied at fixed intervals (n Å 3). One centripetal spike occurred for every three
or four stimuli applied (Fig. 11); 10 09 M 5-HT was then
directly applied to the SRT subchamber, and one centripetal
spike was now recorded for every one or two stimuli applied.
The 5-HT concentration was then increased to 10 08 M, and
two centripetal spikes occurred every time a stimulus was
given. Finally, the 5-HT concentration was increased to 10 07
09-15-98 19:29:19
neupas
LP-Neurophys
Downloaded from http://jn.physiology.org/ by 10.220.32.246 on June 17, 2017
FIG . 8. A: MCC stimulation potentiates contractions of the SRT elicited by motor neuron stimulation. Contractions of
the SRT (middle trace) were elicited by stimulating the SRT motor neuron B66 in bursts (top trace). MCC stimulation
altered parameters of B66-elicited muscle contractions. B: changes in the parameters of B66-elicited SRT contractions affect
activity in B21. When B66 is stimulated at higher frequencies SRT contractions increase in size and contract at a faster rate.
Both effects are seen when SRT contractions are modulated by MCC activity (only the effect on size is apparent at the slow
chart speed shown in A). When parameters of SRT contractions are altered in this manner, afferent activity in neuron B21
is increased.
SEROTONERGIC MODULATION OF MECHANOAFFERENT ACTIVITY
1617
M, and centripetal spikes were no longer recorded. When synaptic transmission was blocked by bathing the buccal ganglion
in 0.51 Ca 2/ ; 41 Mg 2/ seawater (Fig. 11B), 5-HT still increased responsiveness to peripherally applied stimuli. Experiments in Figs. 10 and 11 indicate therefore that 5-HT can affect
the peripheral excitability of B21.
To determine whether MCC activity can affect B21 responsiveness a subthreshold mechanical stimulus was repeatedly
applied before, during, and after MCC stimulation. Mechanical
stimuli that were subthreshold before the MCC was stimulated
became suprathreshold during MCC stimulation (n Å 4).
These experiments were then repeated when the normal ASW
bathing both the buccal and cerebral ganglia was exchanged
for 0.51 Ca 2/ ; 41 Mg 2/ (Fig. 12B). Again MCC stimulation
caused subthreshold stimuli to act as suprathreshold stimuli.
These data show therefore that MCC activity can alter the
sensitivity of B21 to peripherally applied mechanical stimuli.
Note, however, that effects of MCC stimulation are not very
dramatic in the sense that they do not elicit afterdischarges in
B21. Thus, although the MCCs may produce increases in B21’s
sensitivity to a peripheral stimulus while it is actually present,
they are not likely to cause B21’s activity to outlast stimulus
presentation.
DISCUSSION
The MCCs have been well characterized in Aplysia and
other mollusks and have been extensively investigated
/ 9k2d$$oc20 J203-8
within the context of feeding behavior (Berry and Pentreath
1976; Cottrell and Macon 1974; Gerschenfeld and Paupardin-Tritsch 1974; Gerschenfeld et al. 1978; Gillette and
Davis 1977; Granzow and Kater 1977; McCrohan and Audesirk 1987; Weinreich et al. 1973; Weiss and Kupfermann
1976; Weiss et al. 1982). Although previous work has indicated that the MCCs exert effects that are widespread, there
has been no indication that the MCCs have effects on sensory
neurons. Thus C2, a mechanoafferent that provides excitatory drive to the MCCs (Weiss et al. 1986b), is not reciprocally excited by MCC activity (K. Weiss, personal communication). Although cerebral mechanoafferent neurons respond to 5-HT, the physiological source of the serotonergic
input to these cells has not, however, been identified (Rosen
et al. 1989a). This study therefore adds to current conceptualizations of the role of the MCCs in that it demonstrates
that effects of the MCCs are not confined to neuromuscular
systems utilized during feeding behavior. Activity of at least
one group of sensory neurons is modulated by the MCCs.
Previous work in Aplysia has indicated that the MCCs are
silent in quiescent animals but begin to fire when animals
are exposed to food (Kupfermann and Weiss 1982; Weiss et
al. 1978). As animals ingest food the level of MCC activity
remains correlated with the general state of arousal. For
example, initially bite speed is high (indicating that animals
are aroused) (Susswein et al. 1978; Weiss et al. 1982), and
the MCCs fire at relatively high frequencies (Kupfermann
09-15-98 19:29:19
neupas
LP-Neurophys
Downloaded from http://jn.physiology.org/ by 10.220.32.246 on June 17, 2017
FIG . 9. Effects of 5-HT on the SRT are
at least partially mediated by cAMP. A: in
this experiment the SRT was isolated and
incubated in 5-HT solutions for 10 min.
3 *,5 *-Cyclic monophosphate (cAMP) levels are expressed in picomoles of cAMP
per milligram of tissue (wet weight; for
each point n Å 4; SEs are plotted). B: to
determine whether 5-HT increases cAMP
levels in the SRT under conditions where
synaptic transmission cannot occur, the
SRT was incubated for 10 min in 10 06 M
5-HT in Ca 2/ -free ASW. Significant increases in cAMP levels were still observed
(n Å 3; SEs plotted). C: membrane-soluble
cAMP analogue 8-CPT–cAMP produces
5-HT–like effects on the SRT. Left:
stretch-induced contraction of the SRT before application of the cAMP analogue.
Middle and right: in the presence of the
cAMP analogue stretch-induced contractions of the SRT increase in amplitude.
1618
ALEXEEVA, BOROVIKOV, MILLER, ROSEN, AND CROPPER
Downloaded from http://jn.physiology.org/ by 10.220.32.246 on June 17, 2017
FIG . 10. 5-HT exerts inhibitory effects on sensory responses. A and B
are records from the same preparation. The preparation was washed for 30
min between applications of 5-HT (not shown). A1: 10 08 M 5-HT induces
contractions of the SRT (bottom trace) and centripetal spikes in neuron
B21 (top trace). A2: in 10 07 M 5-HT contractions are slightly larger, yet
fewer centripetal spikes are elicited in B21. A3: in 10 06 M 5-HT contractions are still larger, yet centripetal spikes are no longer observed in neuron
B21. B shows the initial part of the washout of 10 06 M 5-HT. As the 5HT was washed out centripetal spikes became apparent in neuron B21
despite the fact that contractions did not initially change in size. This
experiment suggests that 5-HT exerts inhibitory effects on B21 peripheral
processes. These inhibitory effects appear to be apparent at concentrations
that are not yet high enough to produce inhibitory effects on SRT contractile
properties.
/ 9k2d$$oc20 J203-8
FIG . 11. 5-HT increases the sensitivity of B21 to peripherally applied mechanical stimuli. A and B are from the same preparation. Mechanical stimuli
were delivered by means of a minispeaker that had a wooden stick perpendicularly attached to the speaker membrane. Reproducible movements of the membrane were regularly elicited by driving the speaker with a Grass stimulator.
Arrows indicate when mechanical stimuli were applied. The preparation was
washed for 30 min between 5-HT applications (not shown). 5-HT was directly
applied to the SRT subchamber. A1: under control conditions a weak stimulus
elicited centripetal spikes in B21 every 4th or 5th time the stimulus was applied.
In 10 09 and 10 08 M 5-HT response frequency increased. In 10 07 M 5-HT,
inhibitory effects were observed. In B the ASW bathing the ganglion was
replaced with 0.51 Ca 2/ ; 41 Mg 2/ ASW.
and Weiss 1982; Weiss et al. 1978). As a meal progresses,
bite speed and MCC activity decrease (Kupfermann and
Weiss 1982; Susswein et al. 1978; Weiss et al. 1982). Therefore these data and data from other studies, e.g., lesion exper-
09-15-98 19:29:19
neupas
LP-Neurophys
SEROTONERGIC MODULATION OF MECHANOAFFERENT ACTIVITY
1619
iments (Rosen et al. 1989b) and experiments where the
MCCs were stimulated in semiintact feeding preparations
(Weiss et al. 1986a), suggested that the MCCs play a role
in inducing the motivational state of food-induced arousal
in Aplysia.
In Aplysia an essential feature of MCC actions at both
the behavioral and synaptic level is that they are modulatory,
i.e., they are relatively prolonged and their effects are contingent on the occurrence of some other event (e.g., Weiss et
al. 1982). Thus at the behavioral level MCC activity does
not initiate feeding. It does, however, alter responsiveness
to an appropriate stimulus, i.e., food (Weiss et al. 1986a).
Similarly, at the synaptic level, MCC stimulation does not
directly activate neurons. Instead excitatory effects of other
inputs can be enhanced (Weiss et al. 1978). Data obtained
in these experiments are in keeping with this general idea.
Centrally, we observed that, although MCC activity can elicit
a slow depolarization in neuron B21 (i.e., act at site 1 in
Fig. 13), it does not cause B21 to spike. MCC activity can,
however, enhance radula mechanoafferent responsiveness to
/ 9k2d$$oc20 J203-8
depolarization from some other source, e.g., from phasic
inputs associated with elements of the feeding CPG.
Peripherally the MCCs enhance the contractility of the
SRT (i.e., act at site 2 in Fig. 13). This effect will also
be manifested in a phase-dependent manner because it is
dependent on activity of the SRT motor neurons, which fire
rhythmically during ingestive motor programs (Borovikov
et al. 1997). The enhanced contractility of the SRT will
affect radula mechanoafferent activity in two ways. If contractions are large enough, radula mechanoafferent activity
will be elicited in the absence of other mechanical stimulation (Cropper et al. 1996b). If contractions are smaller, centripetal spikes may not be directly elicited, but radula mechanoafferent neurons will be more sensitive to externally applied mechanical stimuli (Cropper et al. 1996b). This
mechanism may therefore serve two functions. It may generate radula mechanoafferent activity or act as a peripheral
way of gating or adjusting radula mechanoafferent gain. In
either case it will be phase dependent.
Other experiments show that the MCCs can act outside
09-15-98 19:29:19
neupas
LP-Neurophys
Downloaded from http://jn.physiology.org/ by 10.220.32.246 on June 17, 2017
FIG . 12. Stimulation of the MCC increases responsiveness of B21 to peripherally applied mechanical stimuli. Arrows
indicate when mechanical stimuli were applied. A and B are from the same preparation. A: a subthreshold mechanical stimulus
did not elicit centripetal spikes in neuron B21 until the MCC was stimulated. B: effects seen in A persisted when the normal
ASW bathing the buccal and cerebral ganglia was replaced with 0.51 Ca 2/ ; 41 Mg 2/ ASW.
1620
ALEXEEVA, BOROVIKOV, MILLER, ROSEN, AND CROPPER
the buccal ganglion, presumably in the periphery, and alter
radula mechanoafferent responsiveness to mechanical stimuli (i.e., act at site 3 in Fig. 13). These types of effects are,
however, not very dramatic in the sense that the MCCs
do not induce afterdischarges in B21. Afterdischarges were
observed as a result of noxious stimulation in other mechanosensory neurons in Aplysia (Clatworthy and Walters 1993).
Because neuronal activation outlasts stimulus presentation
when afterdischarges are observed, this phenomenon would
presumably disrupt the phasic nature of radula mechanoafferent activity. Thus at a cellular level 5-HT may exert effects on peripheral processes of B21 that will produce subtle
increases in B21’s sensitivity to a stimulus without causing
B21 to fire in a manner that is temporally unrelated to the
presence of a stimulus.
In future experiments it will be interesting to characterize
these cellular mechanisms of action in more detail. In particular, it is interesting to note that experiments with exogenous
5-HT indicate that inhibitory effects on the peripheral excitability of B21 are apparent at lower concentrations of 5-HT
than inhibitory effects of 5-HT on contractile properties of
the SRT (cf. Figs. 5 and 11). Although physiologically released 5-HT is not likely to reach concentrations that will
be purely inhibitory, physiological concentrations of 5-HT
may produce inhibitory effects that are masked by excitatory
effects. This type of observation was made in the well-studied accessory radula closer neuromuscular system (e.g.,
Brezina et al. 1994). If this is the case it is interesting that
inhibitory effects of 5-HT appear to be the most pronounced
on the tissue where the most ‘‘control’’ may be needed, i.e.,
on sensory terminals.
The central and peripheral effects of the MCCs on radula
mechanoafferent activity appear therefore to be similar in
that they are both likely to be phase specific. Interestingly,
however, they may be manifested during two antagonistic
phases of behavior. For example, central effects of the MCCs
will be effective when radula mechanoafferents receive de-
/ 9k2d$$oc20 J203-8
09-15-98 19:29:19
neupas
LP-Neurophys
Downloaded from http://jn.physiology.org/ by 10.220.32.246 on June 17, 2017
FIG . 13. Data obtained in this study indicate that the MCCs exert at
least 3 types of effects that modify radula mechanoafferent activity. 1)
They act centrally, i.e., in the buccal ganglion, and increase radula mechanoafferent excitability. 2) They act peripherally and increase SRT contractility. Increases in the size of B66-induced SRT contractions are likely to
indirectly alter radula mechanoafferent activity. When SRT contractions
are increased in amplitude and rise time more centripetal spikes are evoked
in B21. 3) MCCs also act outside the buccal ganglion, presumably in the
periphery, and increase radula mechanoafferent responsiveness to peripherally applied mechanical stimuli.
polarizing input from the CPG, i.e., during the radula closing/retraction phase of ingestive motor programs (Rosen et
al. 1993, 1994). In contrast, the effects of the MCCs on
contractile properties of the SRT are likely to be most effective when SRT motor neurons are active, i.e., during the
opening/protraction phase of ingestive motor programs
(Borovikov et al. 1997).
In this respect radula mechanoafferents appear therefore to
be similar to stretch receptor neurons in lamprey (Grillner et
al. 1984). Lamprey stretch receptors are peripherally excited
when contralateral motor neurons are active (Viana Di Prisco
et al. 1990). Additionally, stretch receptors receive depolarizing
input from the CPG during the phase of behavior in which the
peripheral drive to stretch receptors is decreased, i.e., during
ipsilateral motor neuron activity (Vinay et al. 1996). It was
hypothesized that this arrangement may be analogous to the
efferent control of muscle spindles by g-motor neurons in
higher vertebrates (Sjostrom and Zangger 1976). Thus CPGinduced depolarizations may serve to prolong stretch receptor
sensitivity because they may at least partially compensate for
the decreases in peripheral drive that will occur when the ipsilateral musculature contracts (Vinay et al. 1996).
In lamprey, lateral movements that mimic swimming
movements will efficiently entrain centrally generated motor
programs (e.g., Grillner et al. 1981). It appears therefore
that sensory activity can control phase transitions during
locomotory activity. Presumably this is due to the fact that
there are actually two types of receptor neurons, those that
project ipsilaterally and excite network neurons and those
that project contralaterally and inhibit network neurons (Viana Di Prisco et al. 1990). Possibly, radula mechanoafferents
can act in a similar manner. Only one type of radula mechanoafferent has been described, but, as discussed above, these
neurons 1) make excitatory connections with motor neurons
active during radula closing/retraction and 2) make excitatory connections with an interneuron that inhibits motor
neurons active during radula opening/protraction. Thus radula mechanoafferents may do more than simply prolong radula closing/retraction and delay radula opening/protraction
when bites are converted to bite–swallows. Additionally
they may play a role in causing opening/protraction to end
and closing/retraction to begin.
If this is the case, MCC activity in turn may have dual
effects. Because the MCCs increase SRT contractility they
may increase the number of centripetal spikes that are generated in radula mechanoafferents during opening/protraction
as a result of SRT contractions. In this manner MCC activity
may influence transitions between radula opening/protraction and radula closing/retraction. Consistent with this idea,
MCC activity does increase the rate at which ingestive feeding responses are made (e.g., Weiss et al. 1986a). Second,
during the closing/retraction phase of behavior, MCC activity may increase radula mechanoafferent excitability and
may increase radula mechanoafferent responsiveness to
food. These latter effects may partially underlie increases in
biting strength that occur when animals are food aroused
(Weiss et al. 1978).
In summary, afferent activity can be influenced by modulatory neurotransmitters in many systems (e.g., Billy and
Walters 1989; El Manira et al. 1991b; Mar and Drapeau
SEROTONERGIC MODULATION OF MECHANOAFFERENT ACTIVITY
1996; Pasztor and Bush 1989; Rossi-Durand 1993; Wenning
and Calabrese 1995). To address the physiological significance of this type of regulation, we studied interactions between the MCCs and B21. We show that 5-HT can modulate
B21 activity in several ways; its central excitability can be
increased, its peripheral excitability can be increased, and
contractile properties of the SRT can be enhanced (which
will indirectly enhance B21 activity). Experiments with the
MCCs suggest that this modulation will occur in a phasedependent manner. Previous studies have shown that MCC
activity is likely to contribute importantly to food-induced
arousal. Considerable evidence indicates that this effect is
mediated by central and peripheral actions on motor neurons
and feeding musculature. We now show that activity of a
class of sensory neurons is additionally affected.
Received 18 March 1998; accepted in final form 2 June 1998.
REFERENCES
BERRY, M. S. AND PENTREATH, V. W. Properties of a symmetric pair of
serotonin-containing neurones in the cerebral ganglia of Planorbis. J.
Exp. Biol. 65: 361–380, 1976.
BILLY, A. J. AND WALTERS, E. T. Modulation of mechanosensory threshold
in Aplysia by serotonin, small cardioactive peptideB (SCPB ), FMRFamide, acetylcholine, and dopamine. Neurosci. Lett. 105: 200–204, 1989.
BOROVIKOV, D., ROSEN, S. C., AND CROPPER, E. C. SCP-containing sensory
neurons in Aplysia are peripherally activated as a result of motor neuron
activity. Soc. Neurosci. Abstr. 23: 1045, 1997.
BREZINA, V., EVANS, C. G., AND WEISS, K. R. Activation of K current in
the accessory radula closer muscle of Aplysia californica by neuromodulators that depress its contractions. J. Neurosci. 14: 4412–4432, 1994.
BURROWS, M. Organization of neural networks for the control of posture
and locomotion in an insect. In: Neurons, Networks, and Motor Behavior.
Cambridge, MA: MIT Press, 1997, p. 131–136.
BUSCHGES, A. Role of local nonspiking interneurons in the generation of
rhythmic motor activity in the stick insect. J. Neurobiol. 27: 488–512,
1995.
CLARAC, F. AND CATTAERT, D. Invertebrate presynaptic inhibition and motor control. Exp. Brain Res. 112: 163–180, 1996.
CLATWORTHY, A. L. AND WALTERS, E. T. Rapid amplification and facilitation of mechanosensory discharge in Aplysia by noxious stimulation. J.
Neurophysiol. 70: 1181–1194, 1993.
COTTRELL, G. A. AND MACON, J. B. Synaptic connexions of two symmetrically placed giant serotonin-containing neurones. J. Physiol. (Lond.) 236:
435–464, 1974.
CROPPER, E. C., ALEXEEVA, V., EVANS, C. G., ROSEN, S. C., AND MILLER,
M. W. Modulation of the peripheral activation of the B21/B22 neurons
in Aplysia. Soc. Neurosci. Abstr. 22: 2045, 1996a.
CROPPER, E. C., EVANS, C. G., AND ROSEN, S. C. Multiple mechanisms for
peripheral activation of the peptide-containing radula mechanoafferent
neurons B21 and B22 of Aplysia. J. Neurophysiol. 76: 1344–1351,
1996b.
CROPPER, E. C., KUPFERMANN, I., AND WEISS, K. R. Differential firing patterns of the peptide-containing cholinergic motor neurons B15 and B16
during feeding behavior in Aplysia. Brain Res. 522: 176–179, 1990.
DRUSHEL, R. F., NEUSTADTER, D. M., SHALLENBERGER, L. L., CRAGO, P. E.,
/ 9k2d$$oc20 J203-8
CHIEL, H. J. The kinematics of swallowing in the buccal mass of
Aplysia californica. J. Exp. Biol. 200: 735–752, 1997.
EL MANIRA, A., DI CAPRIO, R. A., CATTAERT, D., AND CLARAC, F. Monosynaptic interjoint reflexes and their central modulation during fictive
locomotion in crayfish. Eur. J. Neurosci. 3: 1219–1231, 1991a.
EL MANIRA, A., ROSSI-DURAND, C., AND CLARAC, F. Serotonin and proctolin modulate the response of a stretch receptor in crayfish. Brain Res.
541: 157–162, 1991b.
FOX, L. E. AND LLOYD, P. E. Serotonin and the small cardioactive peptides
differentially modulate two motor neurons that innervate the same muscle
fibers in Aplysia. J. Neurosci. 17: 6064–6074, 1997.
GARDNER, D. Bilateral symmetry and interneuronal organization in the
buccal ganglia of Aplysia. Science 173: 550–553, 1971.
GERSCHENFELD, H. M., HAMON, M., AND PAUPARDIN-TRITSCH, D. Release
of endogenous serotonin from two identified serotonin-containing neurones and the physiological role of serotonin re-uptake. J. Physiol.
(Lond.) 274: 265–278, 1978.
GERSCHENFELD, H. M. AND PAUPARDIN-TRITSCH, D. On the transmitter
function of 5-hydroxytryptamine at excitatory and inhibitory monosynaptic junctions. J. Physiol. (Lond.) 243: 457–481, 1974.
GILLETTE, R. AND DAVIS, W. J. The role of the metacerebral giant neuron
in the feeding behavior of pleurobranchaea. J. Comp. Physiol. 116: 129–
159, 1977.
GRANZOW, B. AND KATER, S. B. Identified higher-order neurons controlling
the feeding motor program of Helisoma. Neuroscience 2: 1049–1063,
1977.
GRILLNER, S., MCCLELLAN, A., AND PERRET, C. Entrainment of the spinal
pattern generators for swimming by mechanosensitive elements in the
lamprey spinal cord in vitro. Brain Res. 217: 380–386, 1981.
GRILLNER, S., WILLIAMS, T. L., AND LAGERBACK, P. The edge cell, a possible intraspinal mechanoreceptor. Science 223: 500–503, 1984.
KUPFERMANN, I. AND WEISS, K. R. Activity of an identified serotonergic
neuron in free moving Aplysia correlates with behavioral arousal. Brain
Res. 241: 334–337, 1982.
LAURENT, G. Sensory control of locomotion in insects. Curr. Opin. Neurobiol. 1: 601–604, 1991.
LLOYD, P. E., KUPFERMANN, I., AND WEISS, K. R. Evidence for parallel
actions of a molluscan neuropeptide and serotonin in mediating arousal
in Aplysia. Proc. Natl. Acad. Sci. 81: 2934–2937, 1984.
LONGLEY, R. D. AND LONGLEY, A. J. Serotonin immunoreactivity of neurons
in the gastropod Aplysia californica. J. Neurobiol. 17: 339–358, 1986.
LOTSHAW, D. P. AND LLOYD, P. E. Peptidergic and serotonergic facilitation
of a neuromuscular synapse in Aplysia. Brain Res. 526: 81–94, 1990.
MAR, A. AND DRAPEAU, P. Modulation of conduction block in leech mechanosensory neurons. J. Neurosci. 16: 4335–4343, 1996.
MCCROHAN, C. R. AND AUDESIRK, T. E. Initiation, maintenance and modification of patterned buccal motor output by the cerebral giant cells of
Lymnaea stagnalis. Comp. Biochem. Physiol. A Physiol. 87: 969–977,
1987.
MILLER, M. W., ROSEN, S. C., SCHISSEL, S. L., CROPPER, E. C., KUPFERMANN, I., AND WEISS, K. R. A population of SCP-containing neurons in
the buccal ganglion of Aplysia are radula mechanoafferents and receive
excitation of central origin. J. Neurosci. 14: 7008–7023, 1994.
NUSBAUM, M. P., EL MANIRA, A., GOSSARD, J. P., AND ROSSIGNOL, S. Presynaptic mechanisms during rhythmic activity in vertebrates and invertebrates. In: Neurons, Networks, and Motor Behavior. Cambridge, MA:
MIT Press, 1997, p. 237–253.
PASZTOR, V. M. AND BUSH, B.M.H. Primary afferent responses of a crustacean mechanoreceptor are modulated by proctolin, octopamine, and serotonin. J. Neurobiol. 20: 234–254, 1989.
PEARSON, K. G. AND RAMIREZ, J. M. Sensory modulation of pattern-generating circuits. In: Neurons, Networks, and Motor Behavior. Cambridge,
MA: MIT Press, 1997, p. 225–235.
ROSEN, S. C., MILLER, M. W., CROPPER, E. C., AND KUPFERMANN, I. Modulation of outputs of a mechanoafferent neuron by sensory, motor, and
interneuronal elements in the feeding pattern generator network of
Aplysia. Soc. Neurosci. Abstr. 20: 23, 1994.
ROSEN, S. C., MILLER, M. W., WEISS, K. R., AND KUPFERMANN, I. SCPcontaining radula mechanoafferent neurons in the buccal ganglion of
Aplysia: synaptic connectivity of identified cells. Soc. Neurosci. Abstr.
18: 1279, 1992.
ROSEN, S. C., MILLER, M. W., WEISS, K. R., AND KUPFERMANN, I. Different
forms of gating of a peptidergic mechanoafferent neuron by central patAND
09-15-98 19:29:19
neupas
LP-Neurophys
Downloaded from http://jn.physiology.org/ by 10.220.32.246 on June 17, 2017
We thank Drs. Irving Kupfermann and Klaudiusz Weiss for valuable
comments on an earlier draft of this manuscript.
This work was supported by an Irma T. Hirschl Career Scientist Award,
National Institutes of Health Grants MH-01267 (a Research Scientist Development Award), MH-51393, MH-35564, GM-32099, and RR-03051
(RCMI), National Science Foundation Award IBN-9722349, and Department of Defense Instrumentation Grant N00014-93-1380. Some of the
Aplysia used in this study were provided by the Division of Research
Resources Grant RR-10294.
Address for reprint requests: E. C. Cropper, Dept. of Physiology and
Biophysics, Box 1218, 1 Gustave L. Levy Place, New York, NY 10029.
1621
1622
ALEXEEVA, BOROVIKOV, MILLER, ROSEN, AND CROPPER
/ 9k2d$$oc20 J203-8
tryptamine containing neurons from ganglia of Aplysia-californica and
Tritonia-diomedia. J. Neurochem. 20: 969–976, 1973.
WEISS, K. R., CHIEL, H. J., KOCH, U., AND KUPFERMANN, I. Activity of an
identified histaminergic neuron, and its possible role in arousal of feeding
behavior in semi-intact Aplysia. J. Neurosci. 6: 2403–2415, 1986a.
WEISS, K. R., COHEN, J. L., AND KUPFERMANN, I. Modulatory control of
buccal musculature by a serotonergic neuron (metacerebral cell) in
Aplysia. J. Neurophysiol. 41: 181–203, 1978.
WEISS, K. R., KOCH, U. T., KOESTER, J., ROSEN, S. C., AND KUPFERMANN,
I. The role of arousal in modulating feeding behavior of Aplysia: neural
and behavioral studies. In: The Neural Basis of Feeding and Reward.
Brunswick, ME: Haer Institute, 1982, p. 25–57.
WEISS, K. R. AND KUPFERMANN, I. Homology of the giant serotonergic
neurons (metacerebral cells) in Aplysia and pulmonate molluscs. Brain
Res. 117: 33–49, 1976.
WEISS, K. R., MANDELBAUM, D. E., SCHONBERG, M., AND KUPFERMANN, I.
Modulation of buccal muscle contractility by serotonergic metacerebral
cells in Aplysia: evidence for a role of cyclic adenosine monophosphate.
J. Neurophysiol. 42: 791–803, 1979.
WEISS, K. R., SHAPIRO, E., AND KUPFERMANN, I. Modulatory synaptic actions of an identified histaminergic neuron on the serotonergic metacerebral cell of Aplysia. J. Neurosci. 6: 2393–2402, 1986b.
WENNING, A. AND CALABRESE, R. L. An endogenous peptide modulates the
activity of a sensory neurone in the leech hirudo medicinalis. J. Exp.
Biol. 198: 1405–1415, 1995.
WOLF, H. AND BURROWS, M. Proprioceptive sensory neurons of a locust
leg receive rhythmic presynaptic inhibition during walking. J. Neurosci.
15: 5623–5636, 1995.
YEOMAN, M. S., BRIERLEY, M. J., AND BENJAMIN, P. R. Central pattern
generator interneurons are targets for the modulatory serotonergic cerebral giant cells in the feeding system of Lymnaea. J. Neurophysiol. 75:
11–25, 1996.
YEOMAN, M. S., PIENEMAN, A. W., FERGUSON, G. P., TER MAAT, A., AND
BENJAMIN, P. R. Modulatory role for the serotonergic cerebral giant cells
in the feeding system of the snail, Lymnaea. I. Fine wire recording in
the intact animal and pharmacology. J. Neurophysiol. 72: 1357–1371,
1994a.
YEOMAN, M. S., PIENEMAN, A. W., FERGUSON, G. P., TER MAAT, A., AND
BENJAMIN, P. R. Modulatory role for the serotonergic cerebral giant cells
in the feeding system of the snail, Lymnaea. II. Photoinactivation. J.
Neurophysiol. 72: 1372–1382, 1994b.
09-15-98 19:29:19
neupas
LP-Neurophys
Downloaded from http://jn.physiology.org/ by 10.220.32.246 on June 17, 2017
tern generator neurons in the feeding system of Aplysia. Soc. Neurosci.
Abstr. 19: 1700, 1993.
ROSEN, S. C., SUSSWEIN, A. J., CROPPER, E. C., WEISS, K. R., AND KUPFERMANN, I. Selective modulation of spike duration by serotonin and the
neuropeptides, FMRFamide, SCPB , buccalin and myomodulin in different
classes of mechanoafferent neurons in the cerebral ganglion of Aplysia.
J. Neurosci. 9: 390–402, 1989a.
ROSEN, S. C., WEISS, K. R., GOLDSTEIN, R. S., AND KUPFERMANN, I. The
role of a modulatory neuron in feeding and satiation in Aplysia: effects
of lesioning of the serotonergic metacerebral cells. J. Neurosci. 9: 1562–
1578, 1989b.
ROSSI-DURAND, C. Peripheral proprioceptive modulation in crayfish walking leg by serotonin. Brain Res. 632: 1–15, 1993.
SCOTT, M. L., BREZINA, V., AND WEISS, K. R. Ion currents and mechanisms
of modulation in the radula opener muscles of Aplysia. J. Neurophysiol.
78: 2372–2387, 1997.
SILLAR, K. T. Spinal pattern generation and sensory gating mechanisms.
Curr. Opin. Neurobiol. 1: 583–589, 1991.
SILLAR, K. T. AND SKORUPSKI, P. Central input to primary afferent neurons
in crayfish, Pacifastacus leniusculus, is correlated with rhythmic motor
output of thoracic ganglia. J. Neurophysiol. 55: 678–688, 1986.
SKORUPSKI, P. AND SILLAR, K. T. Phase-dependent reversal of reflexes mediated by the thoracocoxal muscle receptor organ in the crayfish, Pacifastacus leniusculus. J. Neurophysiol. 55: 689–695, 1986.
SJOSTROM, A. AND ZANGGER, P. Muscle spindle control during locomotor
movements generated by the deafferented spinal cord. Acta Physiol.
Scand. 97: 281–291, 1976.
SUSSWEIN, K. R., COHEN, J. L., AND KUPFERMANN, I. The effects of food
arousal on the latency of biting in Aplysia. J. Comp. Physiol. 123: 31–
41, 1978.
VIANA DI PRISCO, G., WALLEN, P., AND GRILLNER, S. Synaptic effects of
intraspinal stretch receptor neurons mediating movement-related feedback during locomotion. Brain Res. 530: 161–166, 1990.
VINAY, L., BARTHE, J., AND GRILLNER, S. Central modulation of stretch
receptor neurons during fictive locomotion in lamprey. J. Neurophysiol.
76: 1224–1235, 1996.
WALLEN, P. Spinal networks and sensory feedback in the control of undulatory swimming in lamprey. In: Neurons, Networks, and Motor Behavior.
Cambridge, MA: MIT Press, 1997, p. 75–81.
WEINREICH, D., MCCAMAN, M. W., MCCAMAN, R. E., AND VAUGHN, J. E.
Chemical, enzymatic and ultrastructural characterization of 5-hydroxy-