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The Journal of Neuroscience, January 15, 1999, 19(2):845-858
A Pair of Reciprocally Inhibitory Histaminergic Sensory Neurons
Are Activated within the Same Phase of Ingestive Motor Programs in
Aplysia
Colin G.
Evans1,
Vera
Alexeeva1,
Jurgen
Rybak1,
Tuula
Karhunen1,
Klaudiusz R.
Weiss1, 2, and
Elizabeth C.
Cropper1, 2
1 Department of Physiology and Biophysics and the
2 Fishberg Center for Research in Neurobiology, The Mt.
Sinai Medical Center, New York, New York 10029
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ABSTRACT |
Previous studies have shown that each buccal ganglion in
Aplysia contains two B52 neurons, one in each
hemiganglion. We now show that there are two B52 neurons in a single
buccal hemiganglion and four cells in an animal. We also show that the
B52 neurons are histamine-immunoreactive and use reverse phase
HPLC to show that the histamine-immunoreactive substance is
authentic histamine. Previous studies have shown that the B52 neurons
make numerous inhibitory synaptic connections with neurons active
during the radula closing/retraction phase of ingestive motor
programs. A computational model of the Aplysia feeding
central pattern generator has, therefore, suggested that the B52
neurons play a role in terminating closing/retraction. Consistent with
this idea we show that both B52 neurons fire at the beginning of radula
opening/protraction. We also show that both B52 neurons are sensory
neurons. They are depolarized when a flap of connective tissue adjacent
to the buccal commissural arch is stretched. During ingestive feeding
this is likely to occur at the peak of closing/retraction as
opening/protraction begins. In the course of this study we compare the
two ipsilateral B52 neurons and show that these cells are virtually
indistinguishable; e.g., they use a common neurotransmitter, make the
same synaptic connections, and are both sensory as well as premotor
neurons. Nevertheless we show that the B52 neurons are reciprocally
inhibitory. Our results, therefore, strikingly confirm theoretical
predictions made by others that neurons that inhibit each other will
not necessarily participate in antagonistic phases of behavior.
Key words:
central pattern generator; feeding behavior; proprioceptive input; sensorimotor integration; half-center oscillator; histamine
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INTRODUCTION |
A common feature in central pattern
generating circuits is reciprocal inhibition (Perkel and Mulloney,
1974 ; Getting, 1989; Marder and Calabrese, 1996). Often reciprocally
inhibitory neurons fire in alternating bursts of activity and drive
antagonistic phases of behavior. Recent theoretical studies have,
however, pointed out that this may not always be the case (e.g., Wang
and Rinzel, 1992; Van Vreeswijk et al., 1994; Sharp et al., 1996). For
example, in the most extreme case, reciprocal inhibition can synchronize neurons when the kinetics of the synaptic inhibition is
slow relative to the oscillation period (Wang and Rinzel, 1992; Van
Vreeswijk et al., 1994). In other cases, reciprocally inhibitory neurons may not fire in alternating bursts of activity but may spike in
alternation (Sharp et al., 1996). In a number of systems single spikes
may not be sufficient to drive alternating phases of behavior. Thus,
theoretical studies have suggested that reciprocally inhibitory neurons
will not necessarily drive antagonistic phases of behavior.
Experimental data from this study strikingly confirm this prediction.
The neurons we describe are the B52 neurons in the marine mollusc
Aplysia californica. These cells were originally described as bilaterally symmetrical buccal premotor neurons that are activated during rhythmic motor programs (Plummer and Kirk, 1990). Plummer and
Kirk (1990) characterized the B52 neurons using physiological and
morphological techniques. They found one neuron in each buccal hemiganglion (1) that was located on the rostral surface of the ganglion just lateral and ventral to neuron B51, (2) that showed postinhibitory rebound excitation, and (3) that had processes in a flap
of connective tissue adjacent to the buccal commissural arch. Plummer
and Kirk (1990) also showed that the B52 neurons make inhibitory
synaptic connections with a number of neurons in the ventral motor
neuron cluster and suggested that they may cause an overall shutdown of
patterned activity. More recently, Baxter and coworkers have
characterized additional synaptic connections of the B52 neurons
(Baxter and Byrne, 1991) and generated a computational model of the
Aplysia feeding central pattern generator (CPG) (Baxter et
al., 1997). Computational results more specifically suggest that the
B52 neurons may play a role in terminating the radula closing/retraction phase of ingestive motor programs.
Our experiments now show that there are actually two reciprocally
inhibitory neurons in each buccal hemiganglion that have the
characteristics described by Plummer and Kirk (1990). Thus there are
four B52 neurons in one animal. Additionally, our data further
characterize the B52 neurons in that we show that these cells are
histaminergic mechanoafferents that are both activated at the
beginning of the opening/protraction phase of ingestive motor programs.
These data are consistent with the role of the B52 neurons suggested by
Baxter et al. (1997). Our results, therefore, add to the growing body
of data that suggest that neurons that play a prominent role in pattern
generation can additionally function as sensory neurons (e.g., Pearson
and Ramirez, 1997). We discuss the possible physiological significance
of this arrangement and the possible physiological significance of the
reciprocal inhibition between the B52 neurons.
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MATERIALS AND METHODS |
Animals. Experiments were conducted with 250-350 gm
Aplysia californica that had been maintained in holding
tanks containing 14-16°C artificial seawater (ASW). Animals were
anesthetized by injection of isotonic MgCl2 and then
dissected to create reduced preparations.
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 (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.
Immunocytochemistry. B52 neurons were physiologically
identified and injected with carboxyfluorescein as described above. Ganglia were fixed using a procedure that was similar to one developed by Panula et al. (1988) and modified by Elste et al. (1990).
Specifically, tissue was placed in a solution of 4% carbodiimide in
0.1 M phosphate buffer with 30% sucrose at room
temperature for 2 hr and processed for whole-mount immunocytochemistry
as has been described (Longley and Longley, 1986; Miller et al., 1994).
The primary antiserum (rabbit host; Incstar, Stillwater, MN) was used
at a dilution of 1:100 and was applied for 2-3 d at 4°C. The second
Cy3-conjugated antibody (anti-rabbit IgG heavy and light chain; goat
host; Jackson ImmunoResearch, West Grove, PA) was applied for up
to 3 d (1:100 dilution; 4°C). In some cases ganglia were cleared
with glycerol. In all cases tissues were viewed with a Nikon microscope
equipped with epifluorescence and were photographed with Tri-X
(ASA 400) film. Drawings were made with the aid of a drawing tube.
Electron microscopy. Methods used for electron microscopy
were similar to those of Vilim et al. (1996). Briefly, the tissue was
pinned on Sylgard in a Petri dish and fixed with 4% glutaraldehyde diluted in 0.2 M Na-HEPES buffer, pH 7.6, containing 10%
sucrose and 11 mM magnesium chloride. After thorough
rinsing in buffer, tissue was stained en bloc with buffer containing
1% uranyl acetate for 3 hr and was post-fixed with buffered 1% osmium
tetroxide for 1 hr at room temperature. The tissue was washed with
water, dehydrated in an ethanol series, washed with propylene oxide, infiltrated with EMbed 812, and polymerized at 60°C for 2 d.
Ultrathin sections were cut on Quick Coat-treated 200- 300 mesh
hexagonal nickel grids and counterstained with 1% aqueous uranyl
acetate followed by 0.25% lead citrate. Samples were examined and
photographed with a Zeiss CH-10 electron microscope at 60 kV. Supplies
and reagents were from Electron Microscopy Sciences (Fort Washington, PA).
In situ radiolabeling. In situ radiolabeling
techniques were similar to those used in previous studies (e.g., see
Cropper et al., 1987; Lloyd et al., 1987). Briefly, neurons were
physiologically identified and were marked iontophoretically with fast
green dye (Fisher Scientific, Houston, TX). Buccal ganglia were
incubated for 4 hr in 1 ml of ASW, containing 1 mCi of
[3H]histidine (Amersham, Arlington Heights, IL).
Neurons were individually dissected from labeled ganglia using a
modification of a procedure developed by Ono and McCaman (1980), and
radioactive histamine was extracted in the presence of unlabeled
histamine (Sigma, St. Louis, MO).
Histamine and histidine were separated using HPLC as has been described
by others (e.g., Sondergaard, 1982; Jensen and Marley, 1995).
Specifically, we used a 4.6 × 250 mm reversed-phase column (Microsorb-MB; Rainin, Ridgefield, NJ). Solvent A was 0.1% phosphoric acid (85% H3PO4; Fisher Scientific),
0.006 M NaH2P04 (Fisher
Scientific), and
1% C12H25NaO4S (SDS;
Life Technologies, Gaithersburg, MD); solvent B was 100%
CH3CN (Fisher Scientific). The gradient used was 40-50%
solvent B in 15 min, followed by 50% solvent B for 30 min. The
flow rate was 1.0 ml/min, and samples were collected every 30 sec.
Radioactivity was detected by scintillation counting, and synthetic
histamine and histidine were detected by absorbance measurements
using a V-4 flow spectrophotometer (ISCO Inc., Lincoln, NE) at 215 nm.
Physiological experiments. B52 neurons were identified using
the criteria of Plummer and Kirk (1990). Thus, B52 is located on the
rostral surface of the buccal ganglion, makes monosynaptic inhibitory
connections with B51 and a number of buccal motor neurons, and displays
powerful postinhibitory rebound excitation. Additionally, it has a
distinctive morphology. Its major neurite leaves the buccal ganglion,
enters the buccal commissural arch (Plummer and Kirk, 1990) (Fig.
1), and then arborizes, sending branches
into an adjacent flap of tissue. To characterize the function of this peripheral process, we used a reduced preparation that comprised the
buccal ganglion dissected free from the buccal mass except for a
portion of the I2 muscle. The I2 was sectioned to ensure that
the buccal commissural arch and the adjacent flap of tissue were left
intact. A silk suture (Ethicon, Somerville, NJ) was tied at one end to
the portion of I2 muscle. The other end was left free so that it could
be pulled to stretch the flap of tissue adjacent to the buccal
commissural arch, while recording from neurons.
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Figure 1.
The anatomy of a B52 neuron injected with
carboxyfluorescein dye. B52 has fine ipsilateral processes that can be
observed in the esophageal nerve, buccal nerve 2 (n2),
and buccal nerve 1 (n1) (processes in the ipsilateral
buccal nerve 1 were not observed in this preparation). The major B52
neurite crosses to the contralateral hemiganglion through the buccal
commissural arch. In the contralateral hemiganglion it gives rise to a
dense dendritic field and then enters buccal nerve 1. As the B52
neurite passes through the commissural arch, it gives rise to numerous
smooth, tapering dendrites that branch perpendicularly (indicated by an
asterisk). These branches pass into an adjacent flap of
tissue that emerges from the ventral surface of the buccal ganglia and
anchors it to the I2 muscle (see Fig. 3).
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Experiments that characterized firing patterns of B52 neurons during
motor programs were conducted in a modified version of a preparation
developed by Susswein et al. (1996). This preparation has been
described in detail elsewhere (Evans and Cropper, 1998). Briefly, an
isolated buccal mass with the buccal and cerebral ganglia attached was
pinned to a Sylgard dish. 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. 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. The thread was then pulled through the jaws and attached to an
isotonic force transducer (Harvard Apparatus). 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."
Intracellular recordings were obtained with glass micropipettes filled
with 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.
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 were
obtained from Sigma.
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RESULTS |
The two B52 neurons are histamine-immunoreactive
Two neurons in the ventral motor neuron cluster of each buccal
hemiganglion are histamine-immunoreactive (Elste et al., 1990; Soinila
and Mpitsos, 1991). On the basis of their size and position, we
suspected that at least one of these neurons might be B52, a premotor
neuron originally described by Plummer and Kirk (1990). We, therefore,
identified a B52, based on the description of Plummer and Kirk (1990),
and injected it with carboxyfluorescein dye. Cells identified in this
manner were characterized by histamine-like immunoreactivity. To
identify the second immunoreactive cell, we sought to determine whether
we could identify two neurons with B52-like characteristics in a single
buccal hemiganglion. We did in fact find two such cells. We injected
both cells with carboxyfluorescein to study their anatomy (as described
below) and found that both cells were histamine-immunoreactive (Fig.
2).
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Figure 2.
Physiologically identified B52 neurons are
characterized by histamine-like immunoreactivity. A, B52
neurons were bilaterally injected with carboxyfluorescein dye, and
ganglia were processed for histamine immunocytochemistry.
B, Injected neurons did indeed stain positively. Only
one buccal hemiganglion is shown in each photograph. Fluorescent
processes medial to the injected neurons in the carboxyfluorescein
photograph originate from the contralateral B52 neurons (see Fig. 1).
Scale bar, 50 µm.
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Anatomy of the B52 cells and the flap of tissue that contains the
peripheral processes of the B52 neurons
One B52 neuron is generally located immediately lateral to B51
(Plummer and Kirk, 1990). The second B52 neuron is usually lateral to
the first cell and is generally partly hidden under other neurons. The
two cells, however, have basically similar anatomical features, and we
did not detect any consistent significant anatomical differences
between them (n = 5). They are strikingly multipolar,
extending dense dendritic branching into the neuropile (Fig. 1). Part
of the dendritic field is very superficial, closely applied to the
neurons on the rostral surface of the ganglion, whereas many other
branches project deeply into the neuropile, toward the caudal surface
of the ganglion. We found that B52 had fine ipsilateral processes that
could be observed in the esophageal nerve (observed in the preparation
shown in Fig. 1), in buccal nerve 2 (also observed in the preparation
shown in Fig. 1), and in buccal nerve 1 (not observed in the
preparation shown in Fig. 1). These fine processes were very
superficial and in some cases appeared to travel in the sheath covering
the nerves.
As described by Plummer and Kirk (1990), the major B52 neurite leaves
the ipsilateral buccal hemiganglion and crosses to the contralateral
hemiganglion through the buccal commissural arch (Fig. 1). In the
contralateral hemiganglion, the neurite gives rise to a dense dendritic
field and then leaves the CNS through buccal nerve 1. As the B52
neurite passes through the commissural arch, it gives rise to numerous
smooth, tapering dendrites, which branch perpendicularly, i.e., along
the same axis as the radular sac. These branches pass into what appears
to be an adjacent flap of connective tissue, which emerges from the
ventral surface of the buccal ganglion and anchors the ganglion to the
I2 muscle (Fig. 3). The processes of the
principal neurite are likely to terminate in this flap of tissue
because they were not visualized in the I2 muscle, even when
preparations were kept for 2 d in 10 mM probenecid.
Additionally, fibers with histamine-like immunoreactivity were not
observed on I2. (Although the flap of tissue also contains a blood
vessel that runs from the I2 muscle into the buccal ganglion, B52
processes were never observed in association with this vessel.)
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Figure 3.
A, Drawing of the buccal mass and
the attached buccal ganglion is shown. B, The radula sac
has been moved ventrally to simulate what presumably happens during
feeding behavior in intact animals (Drushel et al., 1997). This
stretched the flap of tissue that contains the processes of B52 (see
Fig. 1). The buccal ganglion was positioned as it is in intact animals;
i.e., only the caudal surface can be seen. For the sake of simplicity
the salivary ducts and cerebral ganglion are not shown. Additionally,
most of the esophagus was removed. CBC, Cerebral buccal
connective; nerve 3, buccal nerve 3.
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To define more precisely the nature of the flap of tissue that contains
the peripheral processes of the B52 neurons, we examined the flap using
electron microscopy. We found fibers and cell types that have
been observed in the fibrous sheath that encloses the Aplysia nervous system (e.g., Coggeshall, 1967). For
example, the bulk of fibers were cylindrical and showed cross-banding
or beading (Fig. 4). These fibers are
presumably collagenous connective tissue fibers (Coggeshall, 1967).
Additionally, we found neural processes that contained dense core
vesicles and cells that morphologically resemble the molluscan muscle
fibers that are observed in the sheath of Aplysia
(Rosenbluth, 1963; Coggeshall, 1967; Prescott and Brightman, 1976)
(Fig. 4). Muscle cells were not found in bundles and generally did not
make contacts with their neighbors as has been described in the
Aplysia sheath (e.g., Prescott and Brightman, 1976).
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Figure 4.
Electron micrograph of the connective tissue flap
that connects the ventral surface of the buccal ganglion to the I2
muscle. Embedded in a collagenous matrix are several processes of
smooth muscle cells (arrows). A wide band of ground
substance (g) and of collagen fibers
(c) separates the cells from each other. Scale
bar, 1 µm.
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The histamine-like-immunoreactive substance is
authentic histamine
Previous investigators have demonstrated that
histamine-immunoreactive neurons take up
[3H]histamine, whereas those that are not
histamine-immunoreactive do not (Elste et al., 1990). In this study we
used in vivo radiolabeling experiments to determine whether
the histamine-like-immunoreactive substance present in the B52 neurons
was in fact authentic histamine (Fig.
5A). Toward this end, B52
neurons were physiologically identified and injected with fast green
dye. Buccal ganglia were incubated in radiolabeled histidine so that
B52 neurons would take up the histidine and synthesize radiolabeled
histamine. Individual cells were then removed from the buccal ganglion
and subjected to RP-HPLC to separate the radiolabeled histidine and
histamine. We were not able to use this technique to detect histamine
in single neurons; consequently chromatography was performed on groups
of eight cells. To evaluate the specificity of this procedure, we
additionally processed B4/B5 neurons from the same buccal hemiganglia.
This procedure was repeated three times; i.e., a total of 24 B52
neurons were processed. In B52 neurons we found that the average
conversion of histidine to histamine was 4.1% (± 1.1). B4/B5 neurons
converted only 0.59% (± 0.18) of the histidine to histamine. We used
a Student's two-tailed t test to determine that this
difference was statistically significant (t = 4.05;
p < 0.01).
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Figure 5.
Biochemical confirmation that B52 neurons do in
fact contain authentic histamine. A, The procedure used
in the experiments. Briefly, B52 neurons were physiologically
identified and injected with fast green dye. (1) Buccal ganglia were
then incubated in a radioactive histamine precursor, i.e.,
[3H]histidine. (2) B52 neurons took up the
precursor and synthesized radiolabeled histamine. (3) Individual
neurons were then removed from buccal ganglia and placed in tubes that
had quantities of synthetic histamine and histidine that are easily
detected with absorbance measurements. (4) Synthetic and native
material was subjected to RP-HPLC. Radiolabeled material that coeluted
with synthetic histidine was identified as native
[3H]histidine, and radiolabeled material that
coeluted with synthetic histamine was identified as native
[3H]histamine. B1,
B2, An experiment in which eight B52 neurons were
chromatographed together. B2 is an enlargement of the
boxed region of the graph in B1. In this
experiment the histidine to histamine conversion was 4.2%. The average
conversion was 4.1% (n = 3).
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The B52s as sensory neurons
We found that the two putative B52 neurons made the same
monosynaptic connections. Most of these connections have been described by Plummer and Kirk (1990) and are with buccal motor neurons (i.e., neurons B3, B9, B15, B16, and B51). One additional connection we
observed was with the neurons B8a and B8b, which are radula closer
motor neurons (Morton and Chiel, 1993) (Fig.
6). Connections between the B52s and
motor neurons are likely to be outputs of the B52s. We sought to
determine, therefore, whether there were also similarities in
"inputs" to the two cells. More specifically we sought to determine
whether both B52 neurons are sensory neurons and, if so, whether the
same type of stimulus activates both cells.
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Figure 6.
The B52 neurons make a synaptic connection with
the B8 neurons. A, Both B52 neurons produce
one-to-one IPSPs in the B8 neurons. The B8 neurons do not
produce synaptic potentials in either B52 neuron. B, The
B52-B8 connection is monosynaptic; i.e., it is observed when ganglia
are placed in high divalent cation solutions.
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That the B52 neurons might be sensory cells was suggested by the fact
that both of these neurons have peripheral processes yet do not appear
to be motor neurons. Thus, the major B52 neurite has branches that
appear to terminate in the connective tissue flap adjacent to the
buccal commissural arch (Figs. 1, 3). When B52 neurons were stimulated,
however, this piece of tissue did not contract. In contrast, excitatory
responses were observed when the connective tissue was stretched
(n = 5). If the B52 was at its resting membrane
potential when the connective tissue was stretched, then action
potentials were generally recorded (Fig. 7A1). If, however, the B52 was
hyperpolarized, subthreshold depolarizing potentials became apparent
(Fig. 7A2, top). Because the connective tissue
flap is very close to the buccal ganglion, we simultaneously recorded
from other buccal neurons to determine whether what appeared to be
depolarizing potentials were actually artifacts caused by movement of
the buccal ganglion. Deflections in recordings in other neurons were
much smaller than were those observed in B52 neurons (e.g., Fig.
7B). To determine whether synaptic transmission was
necessary for depolarizing responses, we replaced the normal ASW
bathing preparations with a 0 Ca+2 and 10 mM Co+2 ASW (which abolishes synaptic
activity in the buccal ganglion). Stretching the flap of connective
tissue still evoked depolarizing responses in B52 neurons (Fig.
7C).
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Figure 7.
The B52 neurons are sensory cells.
A1, Tugging (vertical arrows) on
the connective tissue adjacent to the buccal commissural arch elicited
action potentials in the B52 if the neuron was at its resting membrane
potential. A2, When the B52 was hyperpolarized,
underlying depolarizing potentials became apparent. Although tugs were
clearly effective at evoking responses in B52 neurons
(top), slow pulls (single vertical
arrows) were also effective (bottom).
B, Depolarizing potentials in the B52 did not appear
solely to be movement artifacts because they were much larger than the
deflections recorded from the B52 follower neuron B51. Also note that
when responses were peripherally elicited in B52, postsynaptic
potentials were recorded in B51. The horizontal line
under the B52 trace on the right indicates that current was
injected into the B52 neuron. C, Depolarizing potentials
were not abolished when the normal ASW bathing preparation was replaced
with a solution that abolishes synaptic transmission, i.e., a 0 Ca+2 and 10 mM Co+2
ASW.
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The proximity of the connective tissue flap and the buccal ganglion
made it impossible for us to pull hard enough on the tissue flap to
elicit large depolarizing responses in the B52. We, therefore, used
tugs to perform the above experiments because small changes in membrane
potential were more readily apparent as relatively rapid on-off
responses. Under physiological conditions, however, the connective
tissue is unlikely to be stretched in this manner. We, therefore,
additionally tested slow pulls and found that they also elicited
depolarizations in B52 neurons (Fig. 7A2,
bottom). Interestingly, spikes triggered by peripherally
generated depolarizations were effective at eliciting PSPs in B52
follower neurons (Fig. 7B, left). In fact, these
spikes appeared to be as effective at eliciting PSPs in B52 follower
neurons as were action potentials elicited by injection of current into
the B52 somata (Fig. 7B, right).
The B52 neurons reciprocally inhibit each other
In Aplysia, other pairs of cells that appear to be
indistinguishable from one another have been described, e.g., B4/B5
(Gardner, 1971). In a number of cases these neurons are electrically
coupled. This, however, is not the case for the B52 neurons. Thus, we
found that the B52 neurons monosynaptically inhibited each other
(n = 6; Fig.
8A).
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Figure 8.
A1, The B52 neurons reciprocally
inhibit each other. A2, The connection between the two
B52 neurons appears to be monosynaptic because it is observed in high
divalent cation solutions. B, An excitatory component to
the connection between the two B52s is not revealed when the
postsynaptic B52 is hyperpolarized. Left, The
postsynaptic B52 was at its resting membrane potential.
Middle, right, The postsynaptic B52 was
hyperpolarized. The middle recordings show the
postinhibitory rebound that can be seen in a typical B52 when spiking
is prevented. The postsynaptic B52 on the right was
hyperpolarized so that IPSPs were reduced in size. The postsynaptic
cell has, however, not been hyperpolarized below
EK (note that the chloride IPSPs can still
be observed). Under these conditions a slow synaptic component to the
B52-B52 connection should be apparent if it results from a decreased
potassium conductance. RB52, Right B52; LB52, left B52.
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Plummer and Kirk (1990) showed that B52 neurons generally display
postinhibitory rebound excitation when they are injected with
hyperpolarizing current pulses (they observed this property in 36 out
of 38 preparations) (also see Fig. 8B).
Postinhibitory rebound in the B52 neurons could at least partially
account for the fact that the two cells can function as a half-center
oscillator. Thus, when the two B52 neurons are at, or are close to,
their normal resting potential, they can produce single alternating action potentials (Fig. 9). One B52 fires
producing an IPSP in the second B52. The second B52 rebounds
from the inhibition and generates an action potential that produces an
IPSP in the first B52. The first B52 rebounds, and the cycle repeats.
If the B52s are more depolarized, they can also fire in alternating
bursts of activity (Fig. 10).
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Figure 9.
In otherwise quiescent preparations, the two B52
neurons can fire action potentials in alternation. A,
That the activity of one cell is generated when it rebounds from the
inhibition it receives from its partner neuron is shown when
hyperpolarizing current was injected into the first neuron (indicated
by the horizontal bar). Thus, when the first neuron was
hyperpolarized and could not inhibit the second cell, the second cell
no longer fired. B, The opposite was also true.
Inset, That an inhibitory pulse can initiate activity in
the B52 neurons is shown. At the point indicated by the
horizontal bar, a hyperpolarizing pulse was injected
into a neuron that was not firing spontaneously. The cell rebounded and
generated an action potential.
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Figure 10.
The B52 neurons can fire bursts of action
potentials in alternation. Recordings were obtained from ipsilateral
neurons.
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Although we cannot conclude that all of the rebound activity in the B52
neurons results from the biophysical properties of the postsynaptic
cell, we did not find evidence of a slow excitatory synaptic component
to the B52-B52 connection. Specifically, we sought to determine
whether we could observe a slow excitatory potential attributable to a
decrease in potassium conductance at the B52-B52 synapse. This type of
synaptic effect has been observed previously for histamine in
Aplysia (e.g., Weiss et al., 1986a). When postsynaptic B52
neurons were progressively hyperpolarized as presynaptic B52 neurons
were stimulated, hyperpolarizations reduced the size of IPSPs in
postsynaptic B52s as would be expected (Fig. 8B,
left vs right). When postsynaptic cells were
hyperpolarized in this manner, rebound excitations could obviously no
longer be triggered; i.e., there was no inhibition from which to
rebound (Fig. 8B, right). Under these
conditions a slow excitatory component to the B52-B52 synapse could,
therefore, be "unmasked." We found, however, that although we kept
postsynaptic cells above the potassium equilibrium potential
(approximately  80 mV), excitatory synaptic potentials did not become
apparent (Fig. 8B, middle vs
right).
Activity of B52 neurons during ingestive motor programs
Because the two B52 neurons appear to be indistinguishable yet can
be made to fire out-of-phase in otherwise quiescent preparations, we
sought to determine how they would fire during ingestive motor programs. Ingestive activity was elicited in a modified version of a
preparation developed by Susswein et al. (1996). In these preparations
rhythmic activity is elicited by applying carbachol to the cerebral ganglion.
We found that the two B52 neurons did indeed fire during the same phase
of ingestive motor programs (n = 3; Fig.
11). Spikes evoked in the two B52
neurons generally did not occur exactly simultaneously but did occur at
approximately the same time when feeding motor programs were vigorous.
Specifically, in the section of the recording shown in Figure 11, the
two B52 neurons began to fire before rhythmic activity was actually
generated. At this point the two neurons alternated between firing
more or less in synchrony and alternation (Fig.
12). When motor programs did, however, become rhythmic, the number of spikes in each burst of activity in one
cell was generally the same as the number of spikes in each burst of
activity in the second cell (see numbers above the bursts in Fig. 11). Moreover, 69% of the spikes in one cell occurred within 0.02 sec of a spike in the second cell (the average interspike interval during these bursts of activity is 0.14 sec).
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Figure 11.
The B52 neurons fire during the same phase of
ingestive motor programs. Top, middle,
Intracellular recordings from the B52 neurons. Bottom, A
record from a movement transducer that was attached to the anterior tip
of the radula. Rhythmic activity was elicited by applying carbachol to
the cerebral ganglion before the section of the record shown begins.
Before rhythmic activity was generated, the two B52s alternated between
firing in synchrony and firing in alternation (see Fig. 12).
When rhythmic activity began, the two B52 neurons fired during the same
phase of motor programs, and spikes in the two neurons occurred at
approximately the same time; i.e., 69% of the spikes in one neuron
occurred within 0.02 sec of a spike in the second cell (the average
interspike interval during these bursts of activity was 0.14 sec). The
numbers above the bursts of activity in the
top and middle traces indicate how many
action potentials were in each burst.
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Figure 12.
Data from the experiment shown in Figure 11.
Before rhythmic activity began, the two B52 neurons alternated between
firing in synchrony and firing in alternation. Percent synchrony was
calculated as: 100 [x/(y/2)]. As indicated in the
insets, y is the time between action
potentials in cell #1; x is the time between an action
potential in cell #1 and the next action potential in cell #2. Values
were calculated every time a spike occurred. Cells were firing at ~4
Hz; therefore, a new value was calculated approximately every 0.25 sec.
Forty-three values are plotted. The stretch of recording used to
generate these data covers ~11 sec.
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To determine whether the B52 neurons are active during the
opening/protraction or closing/retraction phases of ingestive motor programs, we simultaneously recorded B52 activity and monitored movements of the buccal mass (Evans and Cropper, 1998)
(n = 6; e.g., see Figs. 11, 13, 14). We found that the
B52 began to spike at the peak of radula retraction. Additionally, we
obtained simultaneous recordings from B52 neurons and neurons that fire
during radula closing/retraction, e.g., neuron B51 (Evans and Cropper,
1998). We found that the B52 neurons fired more or less out-of-phase with these neurons (n = 5; Fig.
13). When feeding motor programs cycled
frequently, the B52 neurons were generally active during the first half
of radula opening/protraction (Fig. 13). When feeding motor programs
began to cycle more slowly, however, there was a pause between peak
radula retraction and the next protraction/opening (as indicated by the
horizontal bars in Fig. 14).
Thus, the radula closed and retracted, opened, and then temporarily
stopped moving before it again protracted and opened. During these
pauses the B52 neurons continued to be active and again fired in
alternation at least part of the time (as they did before rhythmic
activity was elicited; Fig. 11). The possible functional significance
of this prolonged activity in the B52 is discussed below.
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Figure 13.
B52 is active during the opening/protraction
phase of behavior. The top trace is an intracellular
recording from a premotor neuron, B51, that is depolarized and fires
action potentials during the closing/retraction phase of feeding motor
programs (Evans and Cropper, 1998). For the most part, B51 and B52
activity was out-of-phase (see vertical dashed lines).
Also note that depolarizations and action potentials begin to be
recorded in B52 at the peak radula of retraction and continue as the
radula is protracted toward the jaws.
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Figure 14.
B52 activity during a carbachol-elicited motor
program that is not cycling vigorously. Top, An
intracellular recording from a motor neuron that elicits radula opening
is shown (B48) (Evans et al., 1996). Bottom, Starting at
the beginning of the record shown, the radula retracted, opened (see
activity in B48), and paused before the next protraction and opening
were elicited. Middle, B52 continued to fire during
these pauses.
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DISCUSSION |
Possible physiological significance of reciprocal inhibition
between the B52 neurons
We show that there are two ipsilateral B52 neurons. In
Aplysia other well-characterized pairs of neurons that are
virtually indistinguishable are the B4/B5 (Gardner, 1971), B31/B32
(Susswein and Byrne, 1988), B61/B62 (Hurwitz et al., 1994), cerebral
buccal interneuron-8/9 (Xin et al., 1996), and the B8 (Gardner, 1971; Church and Lloyd, 1991; Morton and Chiel, 1993) neurons. In most cases
these pairs of neurons are electrically coupled to each other. In the
case of such interneurons, therefore, this arrangement leads to
feed-forward summation and amplified synaptic output from each neuron
pair (Gardner, 1971).
The B52 neurons, however, are not electrically coupled; they
monosynaptically inhibit each other. At this point we can only speculate as to the functional significance of this arrangement. Others
have used computational techniques to show that reciprocal inhibition
can synchronize neural activity (e.g., Wang and Rinzel, 1992; Van
Vreeswijk et al., 1994). This is not what we observed. During motor
programs that cycle vigorously, the two B52 neurons do fire at similar
frequencies at the beginning of radula opening/protraction. This seems
to result, however, from the fact that the two cells are biophysically
similar to each other and both receive depolarizing input at this
point. IPSPs from partner neurons are presumably not sufficient to
hyperpolarize cells to below-threshold membrane potentials, although
they may contribute to synaptic integration in a subtle way.
Both B52 neurons display powerful postinhibitory rebound excitation.
When reciprocally inhibitory neurons show this phenomenon, stable
oscillatory activity can be elicited (e.g., Satterlie, 1985). This
oscillatory activity can be driven solely by the autoexcitation that
results from the postinhibitory rebound; i.e., oscillatory activity can
occur in the absence of tonic drive from another source and can occur
in the absence of intrinsic oscillating properties of member neurons
(Satterlie, 1985). We show that the B52 neurons are similar in that
they can fire in a reverberatory manner without additional synaptic
input. Previous work has shown, however, that reverberatory activity
can consist of a burst of action potentials in one neuron followed by a
burst of action potentials in an antagonistic neuron. For the B52
neurons, however, reverberatory activity seems to be important within a
single phase of a behavior and consists of a single spike in one cell
followed by a single spike in the second cell. Thus, previous studies
have demonstrated that the autoexcitation that results from
postinhibitory rebound can drive groups of antagonistic interneurons.
We are suggesting that this phenomenon may also play a role in
determining the activity of functionally related neurons within a
single phase of behavior.
Autoexcitation from rebound does not, however, ever seem to be the sole
factor that determines B52 activity, even when motor programs begin to
slow down. This is suggested by the fact that the B52 neurons never
consistently fire in alternation. Instead they begin to alternate
between firing more or less in synchrony and firing in alternation.
Thus, it is likely that autoexcitation plays a role in maintaining B52
activity when synaptic drive is reduced but that, because the B52
neurons must fire in a manner that is appropriate for the ongoing motor
program, synaptic drive can never be completely eliminated.
Physiological role of the B52 neurons
Plummer and Kirk (1990) demonstrated that B52 makes connections
with many feeding neurons. Most of these connections are inhibitory. When B52 is stimulated, therefore, motor programs are not initiated. Plummer and Kirk suggested that B52 is not likely to be a cell that
drives motor patterns but that it is likely to be a premotor neuron
that plays an important role in terminating rhythmic activity. More
recently, Baxter and Byrne (1991) have characterized additional synaptic connections of B52 and have modeled elements of the feeding CPG in Aplysia (Baxter et al., 1997). Baxter et al. (1997)
have more specifically postulated that B52 plays a key role in
terminating radula closing/retraction (Fig.
15). Our data that show that the B52
neurons make an inhibitory synaptic connection with the radula closer
motor neuron B8 and begin to fire as radula opening/protraction begins
are consistent with this idea.
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Figure 15.
A, Computational studies (Baxter
et al., 1997) have suggested that B52 may play a key role in
terminating the closing/retraction phase of ingestive motor programs.
Left, The schematic illustration shows the phase
relationships between depolarization or spiking in the
closing/retraction (C/R) circuitry, depolarization or
spiking in B52, and retractions of the radula. Data from this study
were used to illustrate radula retractions. Thus, as is shown in Figure
13, there is often some overlap between the end of activity in the C/R
circuitry and the beginning of B52 activity. This overlap occurs
approximately at the peak of radula retraction. Here a relatively small
overlap is shown despite the fact that spiking in B51 (a retraction
promoter neuron) can continue for a longer period of time. We show a
relatively small overlap because recent studies have suggested that the
prolonged activity in B51 is centripetally generated, and it has been
demonstrated that central depolarizations can be important to "gate
in" this type of activity (Evans and Cropper, 1998). Figures 11, 13,
and 14 also show that B52 begins to spike when the radula is fully
retracted and that B52 is at least depolarized as the radula protracts.
A, Right, Computational studies indicate
that activity in neurons that are part of the C/R circuitry is
prolonged when simulations are run without including neuron B52 (Baxter
et al., 1997). We hypothesize that radula retractions will also be
enhanced. For comparison, the gray lines in the
diagram on the right indicate the
activity with B52 shown on the left. B,
The schematic illustration shows the phase relationships between
depolarization or spiking in the C/R circuitry,
depolarization or spiking in B52, and retractions of the radula as
described above. Additionally, depolarization or spiking in the radula
opening/protraction (O/P) circuitry has been included. Our
data and those of Drushel et al. (1997) suggest that B52 may be
centripetally activated at the peak of radula retraction (at the point
indicated by the black horizontal line and
arrow). Thus, Drushel et al. (1997) have shown that the
radular stalk moves ventrally at this point. When this occurs, the I2
muscle and the attached connective tissue that contains the terminals
of B52 will be stretched. We show that stretch of this connective
tissue produces depolarizations that can be recorded in the somata of
B52. The schematic illustration of phase relationships shows that this
occurs at a time when enhanced activity in B52 could be important for
terminating enhanced activity in the C/R circuitry.
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In this study we show that centripetal activity is generated in B52
when the connective tissue adjacent to the buccal commissural arch is
stretched. Will this stretch occur during normal behavior? Recent
experiments suggest that it will (Drushel et al., 1997). Thus, Drushel
et al. (1997) have visualized movements of the buccal mass in strongly
illuminated young animals. They have shown that the radular stalk
[also known as the radular sac (e.g., Evans et al., 1996)] becomes
particularly visible when animals ingest food and fully retract the
radula. Under these conditions the radula moves ventrally and produces
a noticeable change in the shape of the buccal mass. When the radula
is in this position, the I2 muscle and the tissue containing the
processes of B52 are likely to be stretched as shown in Figure 3. Data
show, therefore, that the event that peripherally depolarizes B52,
stretch of the connective tissue that contains its processes, is likely
to occur during feeding (Drushel et al., 1997). Specifically, it is
likely to occur when the radula is maximally retracted.
The B52 neurons appear, therefore, to be similar to another
well-studied neuron in Aplysia, C2. Both cells are
histaminergic sensory neurons (e.g., for data on C2, see Ono and
McCaman, 1980; McCaman and Weinreich, 1982; Weiss et al., 1986c).
Additionally, although C2 can function as a mechanoafferent that is
activated when a mechanical stimulus is applied to the perioral zone
(Weiss et al., 1986c), it can also be active in the absence of an
exteroceptive stimulus. Thus, C2 will continue to fire during rhythmic
buccal mass movements even when the stimulus used to trigger feeding movements has been removed (Weiss et al., 1986b). Thus both neurons appear to be capable of functioning as proprioceptors. Despite these
similarities, it does not appear that all proprioceptive or
mechanoafferent neurons in Aplysia are histaminergic. For
example, neuron B21, which is a dual function radula mechanoafferent
(Rosen et al., 1992)/proprioceptive (Cropper et al., 1996) neuron, is glutamatergic (Klein et al., 1998) and is not
histamine-immunoreactive.
Our data indicate that peripheral stretch of the processes of the B52
is likely to contribute to the central activation of this neuron. We
show that when we stretch the connective tissue adjacent to the buccal
commissural arch, depolarizations are recorded from the soma of the B52
indicating that the length constant of the B52 is sufficient to conduct
peripheral information passively to the CNS. These depolarizations are
sufficient to trigger action potentials in the B52 when it is at its
resting membrane potential. Interestingly, this centripetal activity is
also effective at eliciting PSPs in the follower neurons of the B52.
Thus, B52 seems to be unlike some of the other centrally located
feeding sensory neurons in Aplysia in that afferent activity
in B52 does not have to be gated in by a central depolarization
to activate follower cells [compare B52 with B21 (Rosen et al., 1993,
1994) and B51 (Evans and Cropper, 1998)].
Considered in the context of the Baxter model (Baxter et al., 1997),
these data suggest the following: B52 is likely to receive additional
depolarizing input from the periphery at a time when it may play a role
in terminating activity in the radula closing/retraction circuitry
(Fig. 15). This input will be particularly pronounced when
closing/retractions are enhanced and the connective tissue containing
the processes of the B52 is more vigorously stretched. The more
vigorous stretch of its processes will more strongly depolarize B52 and
cause it to fire at an increased frequency. Thus, peripheral
depolarizations of B52 may play a key role in terminating activity in
the closing/retraction circuitry when this phase of behavior is enhanced.
Our data and other studies that have investigated relationships between
sensory neurons and CPGs (Pearson and Ramirez, 1997) have, therefore,
begun to determine how rhythmic behaviors that are generated by
relatively simple CPGs are still able to adjust efficiently to changes
in the external environment. Neurons that are important for basic
rhythm generation can be sensory neurons. When this occurs, changes in
the external environment can immediately produce changes in motor
programs. Motor programs do not need to be modified by complex
reflexes. This type of on-line monitor can be important for changes in
motor programs that are directly elicited by the stimulus that causes
the behavioral change (Pearson and Ramirez, 1997). This study
demonstrates that this type of phenomenon may, however, also be
important when motor programs have to be adjusted in a compensatory
manner. Specifically, we suggest that food contacts the radula during
the closing/retraction phase of feeding and immediately activates
sensory neurons [e.g., radula mechanoafferents (Rosen et al., 1993,
1994; Miller et al., 1994)] that enhance the activity of the radula
closing/retraction circuitry. Enhancements of radula closing/retraction
are necessary to insure that food is deposited in the esophagus. When
radula closing/retractions are more vigorous, it is important that
activity in B52 is also more vigorous. This automatically occurs
because enhanced radula retractions result in an increase in the
peripheral depolarization of B52.
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FOOTNOTES |
Received July 15, 1998; revised Oct. 29, 1998; accepted Nov. 3, 1998.
This work was supported by an Irma T. Hirschl Career Scientist award,
by Research Scientist Development Awards MH01267 and MH01427, and by
Public Health Service Grants MH-51393, GM-32009, and MH-50235. Some of
the Aplysia used in this study were provided by the
National Resource for Aplysia of the University of Miami under Grant RR-10294 from the National Center for Research Resources, National Institutes of Health. We thank Hazel Cropper for the drawings
of the buccal mass.
Correspondence should be addressed to Dr. Elizabeth C. Cropper,
Department of Physiology and Biophysics, Box 1218, 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.
Dr. Rybak's present address: Department of Psychology, Dalhousie
University, Life Science Center, Nova Scotia B3H 4J1, Canada.
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Copyright © 1999 Society for Neuroscience 0270-6474/99/192845-14$05.00/0
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Top
Abstract
Introduction
Compound via MeSH
