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The Journal of Neuroscience, October 1, 1998, 18(19):8016-8031
Proprioceptive Input to Feeding Motor Programs in
Aplysia
Colin G.
Evans1 and
Elizabeth C.
Cropper1, 2
1 Department of Physiology and Biophysics and
2 The Fishberg Center for Research in Neurobiology, The
Mount Sinai Medical Center, New York, New York 10029
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ABSTRACT |
Although central pattern generators (CPGs) can produce rhythmic
activity in isolation, it is now generally accepted that under physiological conditions information from the external and internal environment is incorporated into CPG-induced motor programs.
Experimentally advantageous invertebrate preparations may be
particularly useful for studies that seek to characterize the cellular
mechanisms that make this possible. In these experiments, we study
sensorimotor integration in the feeding circuitry of the mollusc
Aplysia. We show that a premotor neuron with plateau
properties, B51, is important for generating the radula
closing/retraction phase of ingestive motor programs. When B51 is
depolarized in semi-intact preparations, radula closing/retractions are
enhanced. When B51 is hyperpolarized, radula closing/retractions are
reduced in size. In addition to being important as a premotor
interneuron, B51 is also a sensory neuron that is activated when the
feeding apparatus, the radula, rotates backward. The number of
centripetal spikes in B51 is increased if the resistance to backward
rotation is increased. Thus, B51 is a proprioceptor that is likely to
be part of a feedback loop that insures that food will be moved into
the buccal cavity when difficulty is encountered. Our data suggest,
therefore, that Aplysia are able to adjust feeding motor
programs to accommodate the specific qualities of the food ingested
because at least one of the neurons that generates the basic ingestive
motor program also serves as an on-line monitor of the success of
radula movements.
Key words:
proprioceptive input; Aplysia; central pattern
generator; load compensation; plateau potentials; feeding behavior
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INTRODUCTION |
Many rhythmic behaviors are
generated by circuits of neurons referred to as central pattern
generators (CPGs) (Delcomyn, 1980 ). Although CPGs can produce motor
patterns in isolation, it is now generally accepted that sensory
neurons, e.g., proprioceptors, play an important role in generating
many physiological motor programs (e.g., Pearson, 1987).
Proprioceptor-CPG interactions may influence phase transitions and/or
play a role in determining the magnitude of the motor output (e.g.,
Pearson and Ramirez, 1997). The magnitude of the motor output must
increase accordingly when the load on a muscle is increased. This
presumably increases activity in proprioceptors, which directly or
indirectly excite the motor neurons that innervate the loaded muscle.
One mammalian behavior in which this type of compensation has been
extensively studied is locomotion. For example, extensor activity is
increased when an animal walks up an incline. It is likely that this
results, at least in part, from the increased load on extensor muscles and the corresponding increased activity of extensor proprioceptors (Pearson and Ramirez, 1997). Although it has been estimated that peripheral afferents make a significant contribution to locomotion under physiological conditions (e.g., Yang et al., 1991), it has been
difficult to establish this directly in vertebrates.
Proprioceptive input is also important for rhythmic behavior in
invertebrates. Invertebrate preparations have experimentally advantageous features that have made it possible to record from neurons
in intact animals engaged in normal behavior (e.g., Wolf and Pearson,
1988). This work has more clearly established that proprioceptor-CPG
interactions are in fact important under physiological conditions.
Although this has been most extensively documented for arthropods, it
is also likely to be true in molluscs (e.g., Jahan-Parwar et al.,
1983). Proprioceptor-CPG interactions have, however, been less
extensively investigated in molluscan preparations, despite the fact
that the molluscan nervous system is likely to be particularly
advantageous for studies of current interest in this field, i.e.,
studies that characterize synaptic mechanisms important for
sensorimotor integration. For example, sensory neurons in molluscs can
be centrally located and accessible for electrophysiological manipulation and biochemical characterization (e.g., Rosen et al.,
1992, 1993, 1994; Miller et al., 1994; Cropper et al., 1996).
In these experiments, conducted in the feeding circuitry of
Aplysia, we demonstrate that a centrally located premotor
neuron with plateau properties, B51 (Plummer and Kirk, 1990), is
a proprioceptor. We characterize the peripheral mechanism that
generates centripetal activity in B51 and demonstrate that centripetal
spikes in B51 have to coincide with a rhythmic central depolarization
if they are to be effective at driving follower neurons. We also show that centripetal spikes are not generated in B51 during all feeding motor programs. They are particularly apparent during swallowing-like responses when an object is moved into the buccal mass. Finally, we
provide evidence that changes in B51 activity are in fact likely to
have an impact on radula movements. Thus, B51 is not only a proprioceptor but also an interneuron whose activity strongly impacts
buccal motor programs.
Parts of this paper have been published previously (Evans and Cropper,
1997).
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MATERIALS AND METHODS |
Animals. Aplysia californica
(200-400 gm) were maintained at 14-16°C in 150 gallon holding tanks
containing aerated artificial seawater (ASW). In all experiments,
animals were anesthetized with isotonic magnesium chloride (50%
w/v).
Identification of neurons. B51 was initially identified as
described by Plummer and Kirk (1990); i.e., it has plateau properties, and a number of its synaptic connections have been characterized. In
addition, we routinely filled B51 with 5(6)-carboxyfluorescein dye (see
below) to verify its anatomy and characterized additional synaptic
connections that are described in this report.
B44 was originally identified in an isolated buccal mass preparation in
which a cut was made through the pharynx to expose the radula halves.
The esophageal nerves and buccal nerve 1 were cut, and the buccal
ganglion was twisted slightly to expose its caudorostral surface. B44
was identified using three criteria. (1) When it was depolarized, it
caused a bilateral contraction of the inner I4 leaflets and a
backward rotation with slight opening of the radula. (2) This
contraction evoked centripetal spikes in B51. (3) B44 was filled with
carboxyfluorescein, and its anatomy was characterized. The neuron
identified in this manner was then confirmed as being the cell
described by Church and Lloyd (1994) (P. Church, personal
communication).
B8 motor neurons were identified on the basis of morphology [as
determined by injection of carboxyfluorescein (see below)] and
position and their ability to cause the radula to close (Gardner, 1971;
Church and Lloyd, 1991; Morton and Chiel, 1993b).
Characterization of the innervation of the I4 muscle
complex. An isolated buccal mass with the buccal ganglion attached
was pinned ventral side up in a Sylgard-lined dish. The buccal ganglion was pinned to expose the caudorostral surface, and the I2 muscle was
trimmed to expose the I4 muscle. A cut was then made from the jaws,
splitting I6 so that the inner leaflets of the I4 muscles were exposed.
This permitted access for intracellular recording from muscle
fibers.
To measure length changes of the I4 muscles, we reduced the buccal mass
further so that it consisted of the radula sac, the I4 muscles, and the
intact radular nerve. Contractions were transduced using previously
described methods (Evans et al., 1996). Briefly, a wooden beam was
attached at its midpoint to the rotating arm of a semi-isotonic force
transducer (the transducer shown in Fig. 1; Harvard Apparatus), which served as a
movement detector. One end had a metal hook to which the freed end of
the I4 muscle was attached by means of a silk suture. The other half of
the beam was marked with a centimeter scale, along which a known weight could be moved to vary the load on the muscle. A stop was placed under
the wooden beam to control muscle length.
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Figure 1.
Schematic illustration of the preparation used to
transduce movements of the buccal mass. (Ganglia are not shown.) A
string was tied to the anterior tip of the radula and attached
to a movement transducer. This transducer detects movement of the
radula toward the jaws, which is referred to as protraction
(arrow 1), and movement of the radula toward esophageal
tissue, which is referred to as retraction (arrow 2).
Protraction produces a downward deflection in transducer records, and
retraction produces an upward movement. The rest position of the radula
(the position before movement is elicited) is indicated by the
dotted line in the bottom diagram.
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In experiments in which synaptic transmission in the nervous system was
selectively abolished, the buccal ganglion was encircled by a ring of
Vaseline, which "sealed" a Lucite subchamber in place. The ASW
bathing the buccal ganglion could then be specifically exchanged while
the periphery remained in normal ASW.
The carbachol preparation. These experiments were conducted
in a modified version of a preparation developed by Susswein et al.
(1996). Specifically, an isolated buccal mass with the buccal and
cerebral ganglia attached was pinned to a Sylgard dish. Two pins were
inserted through the jaws on either side, to prevent them from closing.
No other pins pierced the buccal mass, although a pin was pushed into
the Sylgard on either side of the buccal mass to prevent it from
rolling during motor programs. All buccal nerves were intact except for
the esophageal nerve. The buccal and cerebral ganglia were pinned to a
raised Sylgard platform, and the cerebral ganglion was isolated in a
Lucite chamber sealed onto the Sylgard with Vaseline. The esophagus was
removed, and a hole was cut in the pharynx to expose the radula halves.
Motor programs were elicited by applying 10 3
M carbachol directly to the cerebral ganglion, which was
not desheathed.
To monitor movements of the buccal mass, we tied a silk suture to the
anterior tip of the radula (Fig. 1). The thread was then pulled through
the jaws and attached to the semi-isotonic force transducer described
above (Harvard Apparatus). In these experiments this transducer
detected movement of the radula toward the jaws, referred to as
"protraction," and movement of the radula back toward esophageal
tissue, referred to as "retraction." Protractions and
retractions actually consist of at least two components-forward or
backward rotation and protraction or retraction. We use the terms
protraction and retraction for simplicity because a detailed analysis
of buccal mass movements is beyond the scope of this study (instead see
Drushel et al., 1997). As shown in Figure 1, protraction produces a
downward deflection in transducer records, and retraction produces an
upward movement. The "rest" position of the radula (the position
before movement is elicited) is indicated by a dotted
line.
Recording. Intracellular electrodes were glass micropipettes
filled with a solution of 3 M potassium acetate containing
30 mM KCl. For simultaneous current injection and voltage
recording, we used double-barreled electrodes beveled so that they had
resistances ranging from 5 to 10 M . Recordings from muscle fibers
were made with single-barreled electrodes with resistances of 10-25
M. Preparations were routinely grounded by the use of a chlorided silver wire.
Intracellular dye injection. A 3% solution of the
fluorescent dye 5(6)-carboxyfluorescein in 0.1 M potassium
citrate, titrated to pH 8.0, was iontophoresed into neurons from
single-barreled glass microelectrodes with tips beveled to lower
impedances to ~10 M (see Rao et al., 1986). To reduce active
transport of the dye, probenecid (10 mM final
concentration) was added to preparations (Steinberg et al., 1987; Rosen
et al., 1991), which were kept at 4°C for 12-15 hr.
Reagents. The ASW used in these experiments had the
following composition (in mM): 460 NaCl, 10 KCl, 11 CaCl2, 55 MgCl2, and 5 NaHCO3. All salts and carboxyfluorescein were obtained from Sigma (St. Louis, MO).
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RESULTS |
B51 as a sensory neuron
In their original description of its morphology, Plummer and Kirk
(1990) noted that B51 has a process in the radular nerve. To visualize
this process, we injected B51 with carboxyfluorescein dye
(n = 10). We found that the axon of B51 remains in the
ipsilateral branch of the radular nerve when it divides (Fig.
2A). From this branch,
the axon enters the immediately adjacent tissue of the pharynx/esophagus and can be traced to the black tissue contiguous with
the esophagus that overlies the I4 muscle complex (Fig.
2B)
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Figure 2.
Anatomy of B51. A, Camera lucida
drawing of two B51 neurons in the same preparation filled with
carboxyfluorescein dye. B, Semischematic drawing showing
the peripheral projection of a B51 neuron. Note that the B51 process
can be traced to the black tissue contiguous with
the esophagus overlying the I4 muscle complex.
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The peripheral branch of B51 does not appear to provide efferent
innervation of the I4 muscle. When B51 is stimulated, junctional potentials are not recorded from I4 fibers in the vicinity of the B51
axon. In addition, stimulation of B51 does not elicit discrete,
localized contractions of buccal musculature. In the absence of
evidence that B51 is a motor neuron, we sought to determine whether it
is a sensory neuron. We stretched leaflets of the I4 muscle and found
that a series of 20-25 mV potentials, resembling centripetal spikes,
was recorded from B51 (Fig. 3).
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Figure 3.
Brief stretches applied to each I4 leaflet
(dorsal, medial, and lateral; see Fig. 4B1) evoke
what appear to be centripetal spikes in B51.
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Innervation of the I4 muscles
To determine whether physiological contractions of the I4 muscle
would activate B51, it was necessary to identify the I4 motor neurons.
The I4 muscle complex is not a solid muscle but has a leaflet structure
(Scott et al., 1991). More specifically, we found that the I4 complex
actually consists of outer leaflets and a set of three inner leaflets
(dorsal, medial and ventral) (Figs.
4B1,
5A). The inner leaflets attach
along the length of the posterior of the radula sac, partly fusing with
each other, and attach anteriorly to the I1/I3 muscle block. The inner
leaflets do not attach to esophageal tissue. The inner leaflets are
partly overlain by the outer leaflets. The outer leaflets are shorter and thinner than are the inner leaflets and are attached anteriorly to
the inner leaflets and posteriorly to esophageal tissue (except for a
narrow ventral strip that attaches to the radula sac). Not surprisingly, in view of its gross structural complexity, the I4 muscle
complex is innervated by more than one motor neuron. In this study, we
concentrated on two neurons (B44 and B8) because they are particularly
effective at inducing centripetal activity in B51.
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Figure 4.
B44 innervates the inner leaflets of the I4 muscle
complex. A, Camera lucida drawing of B44 is shown.
Bn 2 and Bn 3, Buccal nerves 2 and
3. B1, Semischematic drawing of the radula and
attached musculature is shown. The I4 muscle complex has been flipped
back to expose the inner leaflets. B2, Intracellular
stimulation of B44 (bottom) elicits EJPs recorded
intracellularly from a fiber of the dorsal inner leaflet
(top). More specifically, EJPs were recorded from the
posterior side of the dorsal leaflet, i.e., the side that is closest to
the outer leaflets. B3, Stimulation of B44
(bottom) also elicits EJPs that can be recorded from the
anterior face of the inner leaflets (top), i.e., the
side shown in B1. Note that EJPs recorded from the
posterior face of the inner leaflets are larger than those recorded
from the anterior face. The posterior face of the inner leaflets lies
directly under the tissue that contains processes of B51.
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Figure 5.
B8 innervates the outer leaflets of the I4 muscle
complex. A, Semischematic drawing of the radula and some
of its attached musculature, including the I4 muscle complex, is shown.
The outer leaflets of the I4 complex are visible because they overlie
the inner leaflets. B, Intracellular stimulation of B8
(bottom) elicits EJPs recorded intracellularly from
fibers of the outer leaflets (top).
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Neuron B44
Neuron B44 is a large (200-300 µm) cell that occupies a
slightly off-center position on the caudorostral surface of the buccal ganglion (Church and Lloyd, 1994) (Fig. 4A). It has
two axons, one projecting branches into the ipsilateral buccal nerves 2 and 3 and the other sending a bifurcating branch into the radular nerve
before crossing the commissure to send further branches along
contralateral nerves 2 and 3. We were not able to unequivocally identify B44 neurons in both buccal hemiganglia in the same
preparation. Given that B44 has bilateral projections, there may indeed
only be one cell per ganglion.
When B44 is stimulated, spikes are one-to-one with excitatory
junctional potentials (EJPs) in each of the ipsilateral and contralateral inner leaflets of the I4 muscle (n = 7;
Fig. 4B2, B3) but not in the outer
leaflets (n = 6). B44 appears to innervate the
posterior aspect of the inner leaflets more strongly than the anterior
aspect because posterior EJPs are larger than anterior EJPs (Fig.
4B2 vs B3). Interestingly, the posterior
I4 leaflets are closer to the tissue in which B51 processes are
visualized. Contractions of the I4 muscle evoked by B44 persist when
the buccal ganglion is placed in a separate subchamber and bathed in 0 Ca+2 and 10 mM Co+2
ASW to block synaptic transmission (n = 5). Although
this does not eliminate polysynaptic transmission in the periphery, we
found that spikes in B44 and EJPs in I4 muscle fibers are reliably
one-to-one, with constant delays of 80-100 msec. Contractions of the
I4 muscles are elicited by B44 when buccal nerves 2 and 3 are cut,
indicating that the axon in the radular nerve innervates this muscle
group. (Spikes in B44 also elicit EJPs in other muscles of the buccal mass, e.g., I1. We did not, however, characterize this innervation in
more detail because we were interested in neuromuscular units that
would activate B51.)
The concerted muscle contractions produced when B44 is stimulated in
intact buccal mass preparations evoke two movements, a backward
rotation of the radula and a slight opening of the posterior halves of
the radula. This opening was not as vigorous as that elicited by B48,
the motor neuron that innervates the I7-I10 muscles (Evans et al.,
1996). Additionally, B48-induced radula openings are accompanied by
radula protraction (instead of retraction) (Evans et al., 1996).
I4 muscle contractions could be produced by stimulating B44 neurons at
frequencies that are observed during rhythmic motor programs. These
frequencies are, however, only reached after the radula
closing/retraction phase of ingestive motor programs has already begun
(e.g., see Fig. 8A in which contractions are elicited when B44 is stimulated at 10-15 Hz vs Fig. 16 in which B44 eventually fires at ~12 Hz during the closing/retraction phase of a
carbachol-elicited motor program). This observation, taken together
with the fact that other motor neurons that are likely to produce
radula retraction have been described (e.g., see Church and Lloyd,
1994), suggests that B44 contributes to radula retraction but is not
solely responsible for it.
Neuron B8
The B8 motor neurons, B8a and B8b, are located on the rostral
surface of the buccal ganglion (Gardner, 1971) and elicit radula closing (Morton and Chiel, 1993b). When the B8 neurons are stimulated, spikes are one-to-one with EJPs in fibers of the outer I4 leaflets of
the I4 muscle (n = 5; Fig. 5B) but not in
those of the inner leaflets (n = 5). Cutting the outer
leaflets of the I4 muscles does not abolish radula closure induced by
B8, so another as yet unidentified muscle group presumably produces
this movement.
Central interactions between B51 and the I4 motor neurons and
centripetal spikes in B51 as a result of I4 muscle contractions
After we had identified the I4 motor neurons, we characterized
central interactions between B51 and these cells and determined whether
motor neuron-induced muscle contractions would elicit centripetal
spikes in B51.
Neuron B44
Neurons B51 and B44 are electrically coupled centrally, with the
electrical coupling being more effective in the B51 to B44 direction
than vice versa (n = 3; Fig.
6). This is apparent with hyperpolarization but is particularly clear with depolarizing pulses.
The resting potential of B51 is generally ~15-20 mV below threshold.
Consequently, when B51 is activated by injecting current, action
potentials ride on top of an underlying DC component (see Fig.
6A, arrow). Thus, B51 effectively
depolarizes B44 and causes it to spike. In contrast, at its resting
potential, neuron B44 is closer to threshold than is B51. Consequently,
when B44 is depolarized, it begins to spike and less effectively
depolarizes B51 (Fig. 6B). In experiments in which
B44 is stimulated to produce I4 contractions and centripetal spikes in
B51, the electrical coupling between B44 and B51 is, therefore, often
not very apparent.
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Figure 6.
Experiment done in an isolated buccal ganglion
showing that B44 and B51 are electrically coupled. Current injections
are indicated by horizontal bars. When B51 is
depolarized (A), it is more effective at driving
B44 than vice versa (B). In part this is
attributable to the fact that B51 is further from threshold when it is
at its resting membrane potential. Consequently, when B51 is activated
by injecting current, there is an underlying DC component to evoked
activity (arrow in A).
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When B44 is stimulated in intact buccal mass preparations, 20-25 mV
excitatory potentials are generally recorded from the soma of B51
(n = 21 out of 27 preparations; e.g., Fig.
7). These potentials do in fact occur
with a delay after the onset of an I4 muscle contraction (Fig.
7A1). Moreover, they continue to be evoked after the current
being injected into B44 is switched off. Several types of data confirm
that B44-elicited potentials in B51 are in fact centripetally elicited.
Namely, potentials are decreased in amplitude when B51 is
hyperpolarized (Fig. 7C). Additionally, potentials are
abolished when the buccal nerves are cut (n = 2; Fig.
7A2) but do persist when buccal ganglia are placed in a
separate subchamber and are bathed in 0 Ca+2 and 10 mM Co+2 ASW (n = 5; Fig.
7B) (which abolishes synaptic transmission).
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Figure 7.
When B44 is stimulated, B51 is activated
centripetally. Experiments were conducted in a reduced preparation in
which the I4 muscle was attached to a semi-isotonic force transducer,
which served as a movement detector. A1, When B44 is
stimulated (top), a contraction is elicited in the I4
muscle (middle), and spikes are recorded in B51
(bottom). Note that responses in B51 are not recorded
until a contraction has been elicited and that activity in B51 outlasts
the B44 stimulation. A2, The same preparation is shown
after the buccal nerves have been cut. Note that B44 stimulation no
longer triggers responses in B51. B, An experiment in a
preparation in which the periphery and buccal ganglion are
pharmacologically isolated is shown. In B1 and
B3, the periphery and buccal ganglion are both in normal
ASW. In B2, the ganglion is in a solution that blocks
synaptic transmission. Note that responses in B51 are recorded even
when transmission in the buccal ganglion does not occur.
C, Hyperpolarization reduces the size of spikes evoked
in B51 in response to stimulation of B44 (C2 vs
C1 or C2 vs C3).
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To determine whether the number of centripetal spikes evoked in B51 is
related to parameters of muscle contractions, we conducted experiments
in reduced neuromuscular preparations and increased contraction size by
increasing the firing frequency of B44. The number of centripetal
spikes elicited in B51 also increased (Fig. 8A). In other
experiments, which were performed in semi-intact preparations, we
increased the resistance to backward rotation by tying a string to the
anterior tip of the radula and holding this string as B44 was fired.
The number of centripetal spikes evoked in B51 was also increased by
this manipulation (n = 6 out of 7 preparations; Fig.
8B). This latter phenomenon was further investigated
in the reduced preparation in which I4 contractions could be
transduced. The number of centripetal spikes evoked in B51 was
increased when the counterweight on the transducer was increased
(n = 2; Fig. 8C). Thus, B51 seems to
function as a proprioceptor that detects the load on the I4 muscle.
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Figure 8.
B51 is a proprioceptor. A,
Experiment conducted in a reduced preparation in which the I4 muscle is
attached to a movement transducer. Increases in the firing frequency of
B44 (top) evoke bigger I4 contractions
(bottom) and more centripetal spikes in B51
(middle). B, Experiment conducted in an
intact buccal mass preparation. When the radula is restrained, more
centripetal responses are recorded in B51. C, Experiment
conducted in a reduced preparation in which the I4 muscle is
counterweighted, i.e., a washer is placed on the free end of the
transducer arm. Increasing the counterweight increases the number of
centripetal spikes in B51 (middle vs left or
middle vs right panels).
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Neuron B8
There is generally no obvious electrical coupling between B51 and
the B8 neurons. When B51 is stimulated, EPSPs are recorded from the
B8s, one-to-one with B51 action potentials (n = 9; Fig. 9A). These EPSPs seem to be
chemical because they are abolished in 0 Ca+2 and 10 mM Co+2 solutions (n = 2; Fig. 9A2). They do not, however, always become larger
when B8 is hyperpolarized, as might be expected for a conventional chemical connection. Interestingly, however, the conductance of B8
increases with hyperpolarization (A. Klein, personal communication). This inward rectification may, therefore, act to reduce the size of
postsynaptic potentials (PSPs) (see Kandel and Tauc, 1966). Thus,
although the B51-B8 connection seems to be chemical in nature, a
definitive characterization of this synapse will not be possible until
the biophysical properties of B8 have been characterized in detail. In
any case, direct connections between the B51 and the B8 neurons only
operate effectively in one direction. When B8 is stimulated in
preparations in which the peripheral musculature is not present, direct
chemical responses are not evoked in B51.
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Figure 9.
A1, Experiment performed in an
isolated buccal ganglion shows that PSPs in B8 are evoked by injection
of current into the soma of B51. A2, PSPs are abolished
when the buccal ganglion is placed in a solution that abolishes
synaptic transmission (i.e., 0 Ca+2 and 10 mM Co+2). B, Experiment
conducted in an intact buccal mass preparation is shown. In this
experiment B8 activity did not elicit centripetal responses in B51
unless an object was placed between the radula halves.
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When B8 is stimulated in semi-intact preparations, however, centripetal
spikes are recorded in B51 in some preparations (i.e., n = 13 out of 22 preparations; Fig. 9B).
Centripetal activity in B51 elicited by stimulating B8 is, however,
generally much less robust than is activity elicited by B44 in that
with repeated trials B8 becomes progressively less effective at evoking
B51 responses. The number of centripetal spikes evoked by B8 can be increased by placing an object between the radula halves and increasing the resistance to radula closing (n = 2 out of 4 preparations; Fig. 9B).
Central gating of afferent activity in B51
Many of the follower neurons of B51 are cells that are
electrically coupled to it (e.g., Fig. 6) (e.g., Plummer and Kirk, 1990). Because centripetal spikes are relatively fast events, they are
obviously attenuated as they are transmitted to coupled cells.
Moreover, because some of the follower neurons of B51 are not close to
threshold when they are at their resting membrane potential (e.g.,
B15), afferent activity in B51 generally does not cause these neurons
to spike. Thus, when B51 is at its resting membrane potential, afferent
activity in B51 is generally not very effective at driving electrically
coupled follower neurons. It is also not very effective at driving B8
(Fig.
10A,C).
As is shown in Figure 9A1, when B51 is activated by
intracellular current injection, PSPs are recorded in B8. Under these
conditions, however, there is a significant underlying depolarization
of the somata of B51. In contrast, however, when B51 is activated
peripherally, PSPs are not recorded in B8 (Fig.
10A,C). Thus, in a quiescent buccal
ganglion, afferent activity in B51 is not likely to effectively drive
its follower neurons.
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Figure 10.
Central depolarization gates afferent activity in
B51. Horizontal bars indicate current injection into
B44. A, Centripetal spikes in B51 do not elicit PSPs in
B8 when B8 is at its resting membrane potential. B,
Current has been injected into the soma of B51, which has been
depolarized by 15 mV. Centripetal spikes now elicit PSPs in B8.
C, B51 is again at its resting membrane potential. The
size of the current pulse injected into B44 is decreased in
B so that the B44 firing frequency will remain
relatively unchanged; i.e., because B51 and B44 are electrically
coupled, some of the current injected into B51 in B is
seen by B44. A smaller current pulse is, therefore, used to drive B44
when B51 is depolarized.
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When B51 is centrally depolarized, however, centripetal spikes do
become effective at driving follower neurons. This is shown in Figure
10, which is an experiment in which centripetal spikes are evoked in
B51 by stimulating B44 at physiological frequencies under two
conditions, i.e., when B51 is at its resting membrane potential (Fig.
10A,C) and when B51 has been
depolarized by 15 mV (Fig. 10B). PSPs are only
recorded in B8 when B51 is centrally depolarized. Obviously this
paradigm could be repeated for each of the other B51 followers.
Because, however, at least eight of these cells have been identified
[i.e., B3, B9, B15, B16, B4, B5, B44, B8 (Plummer and Kirk, 1990;
Figs. 6, 9)] and more are likely to exist, we conducted our next
experiments in a semi-intact buccal mass preparation in which movements
of the buccal mass could be transduced (n = 3). Thus,
with this paradigm, we used movements of the buccal mass as a general
indicator of activity in buccal motor neurons. When B44 stimulation
elicited centripetal spikes in B51 when it was at its resting
potential, movements of the buccal mass were relatively weak (Fig.
11A,
left). (Presumably they were a direct result of the
B44-evoked contraction.) B51 was then depolarized, and centripetal
spikes were again elicited via B44-evoked contractions relatively soon
after the depolarization (Fig. 11A,
middle). Centripetal spikes now triggered a plateau in B51
that elicited spikes in B44 (see bracket in Fig.
11A, middle) and elicited a more vigorous
radula retraction (Fig. 11A, middle, right). In Figure 11B, we performed a
similar experiment except that B51 was held at depolarized potentials
for a longer time before centripetal spikes were triggered. With this
paradigm, it is more obvious that depolarizations alone are not
sufficient to trigger plateaus in B51. Thus, these experiments indicate
that afferent activity in B51 is only likely to be effective at driving follower neurons if B51 is centrally depolarized.
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Figure 11.
A, Experiment conducted in a
semi-intact buccal mass preparation. B44 is depolarized (as indicated
by the horizontal bars), and centripetal spikes are
elicited in B51. When B51 is at its resting potential
(left), these centripetal spikes are not effective at
triggering retractions [as shown by the transducer recording
(bottom trace on the left)]. When B51 is
depolarized (middle, right), however,
afferent activity is effective at triggering retractions (bottom
traces on the middle and right)
and plateau potentials in B51 (middle trace on the
right). The increased activity in B51 is effective at
driving other neurons. Thus, bracketed action potentials
in B44 occur when B44 is no longer being stimulated (top
trace in the middle). B, Similar
experiment except that B51 is depolarized by 20 mV long before afferent
activity is elicited. Although this is a less physiologically relevant
manipulation, inward currents activated by the depolarization itself
are less likely to be changing when afferent activity is elicited.
Under these conditions, depolarization is still effective at
"gating" centripetal activity; i.e., when B51 is depolarized,
afferent spikes trigger activity in B51 that is more long lasting
(bottom trace on right) and is effective
at driving other neurons [again bracketed action
potentials in B44 occur when current is no longer being injected
(top trace on right)].
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B51 is centrally depolarized during ingestive motor programs
To determine whether B51 does in fact receive central input during
ingestive motor programs, we performed experiments in a modified
version of a preparation developed by Susswein et al. (1996). Video
analysis has indicated that programs initiated by applying carbachol to
the cerebral ganglion in these preparations are ingestive [as opposed
to egestive (Susswein et al., 1996)]. Our results confirmed this
because in most cases radula opening and protraction occurred together,
as did radula closing and retraction. [Radula closing occurs during
radula protraction when animals make egestive responses (Morton and
Chiel, 1993a).] Additionally, the accessory radula closer (ARC) motor
neuron B15 was active during carbachol-elicited motor programs
(n = 4; Fig. 12). When intact animals make egestive responses, neuron B15 does not fire (Cropper et al., 1990). We modified the preparation developed by
Susswein et al. (1996) in that we transduced movements of the buccal
mass.
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Figure 12.
Radula movements and intracellular recordings
from B15 and B51 during a carbachol-elicited motor program. The fact
that B15 is active suggests that these programs are ingestive. In
intact animals, B15 does not fire when animals make rejection responses
(Cropper et al., 1990). Left, Activity of B15 and B51
without current injection in either neuron. Right,
Depolarizing current injected into B51. Note that radula retractions
are more vigorous and that activity in B15 remains phase-locked to B51
activity.
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In the majority of preparations (8 out of 15), carbachol induced a
motor program characterized by a weak protraction of the radula from
the rest position toward the jaws and then a retraction back to the
rest position or slightly beyond rest position (e.g., Fig. 12; Fig.
13, middle
panel). This type of activity most closely corresponds to biting-like behavior (as opposed to swallowing-like behavior, the alternative type of ingestive response). Programs are
more biting-like than swallowing-like because the radula does not fully
retract to reach the esophagus. In intact animals, biting occurs when
animals make consummatory responses but do not successfully ingest food
(Kupfermann, 1974). The radula does not fully hyper-retract because
there is no food to be deposited in the esophagus (Fig. 14). We refer to responses as
biting-like because movements of the buccal mass are not identical to
those observed in intact animals. Most noticeably, the radula was never
extended through the jaws as it is in intact animals (Kupfermann, 1974;
Drushel et al., 1997). Protractions shown in Figures 12, 13, 15, and 16
are, therefore, much weaker than they would be in intact animals.
Additionally, it has been hypothesized that retractions during biting
consist of the return of the radula to a neutral position as is shown in Figure 14 (Weiss et al., 1986). Currently this is only a hypothesis; however, even in reduced preparations the radula cannot be easily visualized in the neutral state because it is within the buccal mass.
In our experiments, in which we monitor radula movements, we find that
carbachol commonly elicits a motor program in which the radula retracts
slightly beyond its rest position. Although this might suggest that
current conceptualizations of biting need to be reevaluated, this issue
would obviously be most effectively addressed in experiments in which
radula movements are monitored in intact animals. In summary, we use
biting-like simply as a relative term to contrast biting-like motor
programs with programs in which the radula does retract to reach the
esophagus, referred to as swallowing-like (described below).
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Figure 13.
Radula movements and intracellular recordings
from left and right B51s during a carbachol-elicited motor program. In
this experiment, depolarizing current is periodically injected into
both B51s (e.g., left and right panels).
The B51s are then returned to the resting membrane potential (e.g.,
middle panel). Note that when the B51s are
depolarized by current injection, CPG-elicited depolarizations are also
larger in amplitude and longer in duration. Additionally, radula
retractions become more vigorous. Changes in B51 activity and changes
in radula movements occur in parallel. Thus, when B51 is no longer
depolarized, radula retraction immediately becomes less vigorous. When
B51 is depolarized, again radula movements immediately become
enhanced.
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Figure 14.
Schematic diagram that illustrates current
conceptualizations of how radula movements are thought to change as
food is ingested (Weiss et al., 1986). A, When food is
not ingested, it has been suggested that opening/protraction occurs as
the radula moves from a neutral position to a point where it is
extended through the animal's open jaws (arrow 1). It
has been suggested that the radula then returns to a neutral state
(arrow 2). B, When animals make
swallowing responses, the radula moves from a rest position to a
hyper-retracted position so that food can be deposited in the esophagus
(arrow 1). The radula then returns to the rest position
(arrow 2).
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Simultaneous recordings from left and right B51s revealed that they did
receive a 15-20 mV depolarization during biting-like motor programs
(Figs. 12, left, 13, middle panel). This
depolarization was recorded during the closing/retraction phase of
behavior, which is the phase of behavior in which the I4 muscle complex contracts, and will presumably generate centripetal activity in B51.
Centripetal activity in B51 is more likely to occur during
swallowing-like motor programs than it is during biting-like motor
programs
Although B51 is centrally depolarized during biting-like
motor programs, not many action potentials are recorded from its soma
(Fig. 12, left, 13, middle panel). Thus,
it is likely that little if any centripetal activity is generated under
these conditions (alternatively centripetal activity could be generated
but spikes could be blocked so that they are not apparent in the B51
soma). As described above, carbachol most commonly elicits a
biting-like motor program. In other cases, however, the radula clearly
retracted vigorously beyond the rest position and reached the esophagus when carbachol was applied (n = 4; Fig.
15). We describe these motor
programs as swallowing-like. In other preparations, it was more
difficult to classify movements clearly as biting-like or swallowing-like (n = 3). During swallowing-like motor
programs, spikes are recorded in B51. This is consistent with the idea
that centripetal activity is more likely to be generated in B51 under these conditions.
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Figure 15.
Radula movements and intracellular recordings
from B51 and B8 during a carbachol-elicited motor program in which
radula retractions were relatively vigorous. The B51 neuron was
hyperpolarized during the time indicated by the horizontal
bar. Note that radula retractions immediately became less
vigorous.
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Data obtained in reduced preparations also indicate that centripetal
activity in B51 is increased when the resistance to backward rotation
of the radula is increased (Fig. 8B). Under
physiological conditions, resistance to backward rotation is likely to
be increased when food is ingested. Thus, we might expect that
centripetal activity would be particularly pronounced in B51 during
swallowing responses when an object is pulled into the buccal cavity.
To test this possibility, we elicited biting-like motor programs with
carbachol, and a piece of polyethylene (PE) tubing was placed between
the radula halves as the radula protracted. When the tubing was
successfully grasped between the radula halves and pulled toward the
esophagus (n = 2; Fig.
16), spiking in B51 was indeed enhanced. Although our measurements do not directly indicate whether individual spikes were generated peripherally or centrally in B51, at
least some of the increased activity seems to have been generated
peripherally because it occurs after B51 is no longer centrally
depolarized (see Fig. 16, inset). These findings are, therefore, consistent with the idea that centripetal activity in B51 is
enhanced when an object is pulled into the buccal mass.
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Figure 16.
Radula movements and intracellular recordings
from B51 and B44 during a carbachol-elicited motor program. At the
point indicated by the bar above the top trace, a
piece of PE tubing was inserted between the radula halves. Radula
retraction became more vigorous, and the piece of PE tubing was pulled
through the buccal mass. Activity in B44 and B51 was enhanced in
parallel to changes in radula movements. At least some of the increased
activity in B51 was likely to have been peripherally generated because
spikes are recorded when B51 is no longer centrally depolarized
(arrow in record of B51 activity, in
inset). Also note that spikes in B51 are of different
sizes, which also suggests that they are generated at different
sites.
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Depolarization of B51 enhances radula movements during ingestive
motor programs
We next sought to determine whether changes in the
activity of B51 are in fact likely to have an impact on buccal motor
programs. Thus, although B51 often does not fire at a high frequency
during biting-like or swallowing-like motor programs, it is
electrically coupled to a number of its follower neurons. Consequently,
the magnitude of the rhythmic depolarizations in B51 could play an important role in determining the firing frequency of follower motor
neurons. In a similar vein, the duration of the rhythmic depolarizations in B51 could determine the duration of bursts of
activity in follower motor neurons. Because the feeding muscles that
have been characterized are nonspiking, burst duration generally will
affect both contraction duration and amplitude (e.g., Cohen et al.,
1978; Evans et al., 1996).
To determine whether changes in the activity of B51 alter feeding motor
programs, we elicited rhythmic activity with carbachol and briefly
depolarized or hyperpolarized B51. B51 was depolarized in preparations
in which radula retractions were relatively weak (e.g., Figs. 12, 13)
and hyperpolarized in preparations in which radula movements were more
vigorous (Fig. 15). When B51 was depolarized, activity of other radula
closing/retraction neurons was immediately enhanced, and movements of
the peripheral musculature were immediately changed (n = 8 out of 9 preparations; Fig. 12, 13). Specifically, the radula moved
further beyond the rest position (Figs. 12, 13). These
"hyper-retractions" ceased when the current injection was turned
off. When B51 was hyperpolarized, the opposite was observed; e.g.,
retractions became less vigorous (n = 5 out of 6 preparations; Fig. 15). These data suggest that changes in B51 activity
will produce changes in radula movements during rhythmic motor
programs. They also provide insight into the possible functional
significance of centripetal activity in B51 in that they show that when
activity in B51 is enhanced, as it will be by centripetal activation,
the drive to the radula closing/retraction circuitry will be
enhanced.
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DISCUSSION |
B51 as a pattern-generating interneuron?
The data of Plummer and Kirk (1990) suggested that B51 is part of
the feeding CPG. They showed that B51 receives cyclic input during
spontaneous and nerve-shock-evoked motor programs. Moreover, depolarization of B51 can initiate bursting in many cells, and when
buccal programs cycle spontaneously, depolarization of B51 will
phase-shift activity and entrain it. Because methods for inducing
ingestive motor programs had, however, not been developed, Plummer and
Kirk (1990) could not more precisely define the physiological role of
B51. In this study we addressed this issue. We showed that
depolarization of B51 will produce widespread changes in the activity
of the closing/retraction circuitry during biting-like motor programs.
We additionally studied the role of B51 in a less conventional manner;
i.e., we changed the membrane potential of B51 and measured changes in
radula movements. We showed that depolarization of B51 produces an
enhancement of radula retraction, whereas hyperpolarization of B51
produces a decrease in radula retraction. Thus, B51 seems to play an
important role in generating feeding motor programs. It may be similar
to one of the N2 neurons of Lymnaea (Elliott and Benjamin,
1985; Brierley et al., 1997a,b).
It is unlikely, however, that activity in B51 is necessary for
closing/retraction. In most cases radula movements were not eliminated
when B51 was hyperpolarized. It is possible that hyperpolarizations did
not completely block interactions between B51 and the feeding circuitry. However, cerebral neurons that initiate motor programs synapse directly on both buccal motor neurons and premotor neurons (e.g., Rosen et al., 1991; Xin et al., 1996; Perrins and Weiss, 1998).
Cerebral input to the closing/retraction circuitry is, therefore, not
specifically funneled into B51. Moreover, another closing/retraction
premotor neuron has been identified [i.e., neuron B64 (Hurwitz and
Susswein, 1996)]. Thus, although it is unlikely that radula
closing/retraction is solely driven by B51, our data show that changes
in the activity of B51 can affect this phase of behavior. Changes in
centripetal activity in B51 are, therefore, likely to have an impact on
feeding motor programs.
B51 as a sensory neuron
We show that B51 is a proprioceptor. Although proprioceptors have
been described in the Aplysia feeding circuitry (e.g.,
Jahan-Parwar et al., 1983), their role in behavior has not been
established. For example, B4 and B5 (Jahan-Parwar et al., 1983) make
inhibitory connections with many motor neurons active during radula
closing/retraction (Gardner, 1971; Fiore and Meunier, 1979). It has
been hypothesized, therefore, that B4 and B5 are part of a reflex by
which they are activated when muscles are stretched. When B4 and B5
fire, they would inhibit follower neurons, which would subsequently be
activated as a result of postinhibitory rebound (Jahan-Parwar et al.,
1983). How this reflex could function during motor programs is,
however, unclear. For example, this model implies that B4 and B5 should fire out of phase with inhibited cells. This does not, however, seem to
be the case; B4 and B5 fire during the closing/retraction phase of
ingestive motor programs (e.g., Rosen et al., 1991; Church and Lloyd,
1994). Thus, although previous studies have shown that there are
proprioceptors in Aplysia and other molluscs (e.g., Kater
and Rowell, 1973), other studies have not established that sensory
input is important for the generation of molluscan feeding motor
programs. These results also add to the growing body of data that
indicate that sensory neurons can be intrinsic parts of CPGs (e.g.,
Pearson, 1987; Pearson and Ramirez, 1997).
Central gating of afferent activity in B51
Plummer and Kirk (1990) demonstrated that B51 can generate plateau
potentials. Mechanoreceptors that have complex biophysical properties
have been described (e.g., Combes et al., 1993, 1995, 1997). This type
of arrangement can add to the potential of a sensory neuron for coding
peripheral information if intrinsic activity is generated peripherally.
For example, the anterior gastric receptor of lobster generates bursts
of activity in its dendrites (Combes et al., 1993, 1997). Burst
frequency, burst duration, and intraburst firing frequency can all be
changed by a stimulus (Combes et al., 1997). In contrast, in B51,
intrinsic activity only seems to be elicited centrally. Centripetal
spikes are generated in a straightforward linear manner.
Because B51 does, however, seem to have such a prominent role in
determining the magnitude of motor output, central integration of
information in this cell is likely to be more important. We show that
centripetal activity in B51 is not effective at eliciting responses in
follower neurons unless B51 is simultaneously depolarized centrally.
This type of gating has been described for other sensory neurons in
Aplysia (Rosen et al., 1993, 1994). What specific role the
complex biophysical properties of B51 play in integrating central and
peripheral information is not yet clear. In otherwise quiescent
preparations, we show that when B51 is depolarized by 15-20 mV, as it
is during biting-like motor programs, centripetal spikes can cause B51
to exhibit plateau potentials. Although these potentials may not occur
under physiological conditions, centripetal activity may at least
depolarize B51 to the point at which sustained inward currents are
partially activated. Effects of these currents on synaptic integration
in B51 may be complex (e.g., Simmers and Moulins, 1988a,b).
Physiological role of centripetal activity in B51
When Aplysia do not successfully ingest food, they bite
(Kupfermann, 1974). During biting programs, it is likely that B51 receives a rhythmic central depolarization but is not centripetally activated (Fig. 17). During biting-like
motor programs induced by carbachol, spikes are generally not recorded
in B51. Additionally, the number of centripetal spikes in B51 is
related to the size of I4 muscle contractions and to the degree of
resistance that is encountered as the radula rotates backward. I4
muscle contractions will be smaller when animals bite than when they
swallow because radula retractions are less vigorous. Because food is
not ingested, there also should be little resistance to backward
rotation.
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Figure 17.
Schematic representation of neural activity
during a bite (left) and a swallow
(right). When bites are converted to swallows, activity
in radula closing/retraction motor neurons is enhanced and prolonged
(top). These changes in motor neuron activity result, at
least partially, from the activation of buccal sensory neurons.
Activity is likely to be initiated in radula mechanoafferents at the
end of protraction/opening, as food contacts the maximally extended
radula (middle). Radula mechanoafferents are likely to
remain active as the radula closes on food and may accommodate with
maintained stimulation (Miller et al., 1994). Centripetal activity in
B51 is likely to occur relatively late during closing/retraction
(bottom). Although B44 starts firing as
closing/retraction is initiated, it takes time for the I4 muscle to
contract. Radula mechanoafferents may, therefore, trigger bite to
bite-swallow conversions, whereas centripetal activity in B51 may be
more important as swallows are executed.
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When animals do ingest food and swallow, centripetal activity in B51 is
likely to be initiated (Fig. 16). This is likely to occur because as
food passes through an animal's jaws, it will offer resistance to
backward rotation. What will the function of the increased drive of B51
to the retraction circuitry be? It could actually trigger bite to
bit-swallow conversions. Thus, food of any size could offer enough
resistance to centripetally activate B51, and enhanced B51 activity
could solely produce the changes in the activity of the
closing/retraction circuitry that are observed when animals ingest food
(Cropper et al., 1990). We do not, however, think that this is the case
for several reasons. For example, centripetal activity in B51 is only
likely to occur toward the end of radula closing/retraction (Fig. 17).
B44 starts firing as closing/retraction is initiated, but centripetal
activity in B51 occurs with a delay, because it takes time for the I4
muscle to contract. Centripetal activity in B51 is, therefore, likely to occur too late to trigger bite to bite-swallow conversions (see
Fig. 16 in which enhanced activity in B44 appears to precede spiking in
B51). Moreover, other sensory neurons that are activated by food have
been identified, i.e., the radula mechanoafferents B21 and B22 (Rosen
et al., 1992; Miller et al., 1994). In contrast to B51, centripetal
activity in B21 and B22 is likely to occur early during
closing/retraction. Thus, centripetal activity in B51 may not be
critical for triggering bite to bite-swallow conversions. Instead it
may be important for swallow execution.
Assuming that this is case, an additional question can be asked;
namely, how does B51 activity actually affect the magnitude of radula
retractions when food is ingested? In our carbachol experiments in
which B51 is depolarized, the magnitude of radula retraction is greatly
enhanced. Under these conditions, however, an object cannot be moved
through the buccal mass in a normal manner because the jaws, etc., are
not present and retraction muscles do not have to work against a
physiological load. Although we cannot directly assess the
physiological impact of B51 on the magnitude of radula retraction in
our preparation, data relevant to this issue exist. Namely, experiments
in intact animals have indicated that swallows do occur when food is
counterweighted (within a certain range) but are not increased in
magnitude (Hurwitz and Susswein, 1992). Thus, animals can in fact
overcome difficulties to pull food into the esophagus. Presumably, they
do this by increasing the drive to the retraction circuitry, e.g., by
means of the B51 mechanism. Animals do not, however, increase swallow
magnitude. Increases in the activity of the retraction circuitry are,
therefore, presumably not important because they make retractions
larger than they would have been had resistance not been encountered. Instead they may be important because they allow muscles to cope with
the increased load. This makes sense in that for food to be ingested it
is simply important that it reach the esophagus. Going beyond the
esophagus is not likely to be beneficial. Thus, it is possible that
under physiological conditions, centripetal activity in B51 functions
in a compensatory manner.
In conclusion, B51 is a proprioceptor yet is also a centrally located
neuron whose activity strongly impacts buccal motor programs.
Consequently, ingestive motor programs are likely to be automatically
adjusted to insure that food will be ingested if a reasonable amount of
resistance is encountered.
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FOOTNOTES |
Received April 23, 1998; revised July 15, 1998; accepted July 17, 1998.
This work was supported by an Irma T. Hirschl Career Scientist award,
Research Scientist Development Award MH-01267, and Public Health
Service Grant MH-51393. Some of the Aplysia used in this study were provided by the National Center for Research Resources National Resource for Aplysia at the University of Miami
under National Institutes of Health Grant RR10294. We thank Drs. Paul Church, Itay Hurwitz, and Klaudiusz Weiss for their valuable comments on an earlier draft of this manuscript.
Correspondence should be addressed to Dr. Elizabeth C. Cropper,
Department of Physiology and Biophysics, Box 1218, Mount Sinai School
of Medicine, 1 Gustave L. Levy Place, New York, NY 10029.
Dr. Evans's present address: Phase V Communications Inc., 114 Fifth
Avenue, New York, NY 10011.
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Top
Abstract
Introduction
