1Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York 10029; 2Center for Neurobiology and Behavior, College of Physicians and Surgeons, Columbia University, New York City, New York 10032
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ABSTRACT |
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Hurwitz, Itay, Ray Perrins, Yuanpei Xin, Klaudiusz R. Weiss, and Irving Kupfermann. C-PR neuron of Aplysia has differential effects on "feeding" cerebral interneurons, including myomodulin-positive CBI-12. Head lifting and other aspects of the appetitive central motive state that precedes consummatory feeding movements in Aplysia is promoted by excitation of the C-PR neuron. Food stimuli activate C-PR as well as a small population of cerebral-buccal interneurons (CBIs). We wished to determine if firing of C-PR produced differential effects on the various CBIs or perhaps affected all the CBIs uniformly as might be expected for a neuron involved in producing a broad undifferentiated arousal state. We found that when C-PR was fired, it produced a wide variety of effects on various CBIs. Firing of C-PR evoked excitatory input to a newly identified CBI (CBI-12) the soma of which is located in the M cluster near the previously identified CBI-2. CBI-12 shares certain properties with CBI-2, including a similar morphology and a capacity to drive rhythmic activity of the buccal-ganglion. Unlike CBI-2, CBI-12 exhibits myomodulin immunoreactivity. Furthermore when C-PR is fired, CBI-12 receives a polysynaptic voltage-dependent slow excitation, whereas, CBI-2 receives relatively little input. C-PR also polysynaptically excites other CBIs including CBI-1 and CBI-8/9 but produces inhibition in CBI-3. In addition, firing of C-PR inhibits plateau potentials in CBI-5/6. The data suggest that activity of C-PR may promote the activity of one subset of cerebral-buccal interneurons, perhaps those involved in ingestive behaviors that occur during the head-up posture. C-PR also inhibits some cerebral-buccal interneurons that may be involved in behaviors in which C-PR activity is not required or may even interfere with other feeding behaviors such as rejection or grazing, that occur with the head down.
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INTRODUCTION |
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Complex behavioral acts such as feeding are
typically composed of a number of different but interrelated behavioral
acts. The behavioral sequences that provide the components of each
behavioral act are generated by systems of interconnected neurons
termed pattern generators (PGs) or central pattern generators (CPGs). Studies in several invertebrates suggest that the activity of pattern
generating circuits often is initiated or modulated by the firing of a
small number of higher-order interneurons (Arshavsky et al.
1988, 1989
; Bartos and Nusbaum 1997
;
Delaney and Gelperin 1990
; Frost and Katz
1996
; Kupfermann and Weiss 1978
; Rosen et al. 1991
; Wiersma and Ikeda 1964
). Those
higher-order neurons that exert relatively large effects have been
termed command neurons (Kupfermann and Weiss 1978
;
Wiersma and Ikeda 1964
), command-like neurons
(Deodhar et al. 1994
), or influential neurons
(Arshavsky et al. 1988
). Analogous higher-order neurons,
rather than directly participating in the generation of behaviors,
contribute to the generation of motivational states, which function to
coordinate and optimize the functioning of the various somatic and
visceral behaviors that comprise complex behavioral acts (Teyke
et al. 1990
). An understanding of the interactions between the
various higher-order neurons that control behavior may provide insights into how the nervous system generates decisions about what behavior to
execute and how to modify the chosen behavior based on the specific
conditions of the environment and the internal state of the organism.
In the cerebral-ganglion of Aplysia, an identified neuron,
C-PR, is important in generating a feeding posture (head up) and other
manifestations of an appetitive arousal motivational state associated
with feeding behavior (Nagahama et al. 1994;
Teyke et al. 1990
, 1991
). Consummatory feeding
behaviors, such as biting, are generated by neurons primarily located
in the buccal ganglion, and this circuitry is regulated by a population
of approximately 12 bilateral cerebral-buccal interneurons (CBIs) that
are located in the cerebral ganglion and that project to the buccal
ganglion (Church and Lloyd 1994
; Perrins and
Weiss 1998
; Rosen et al. 1991
; Xin et al.
1999
). The CBIs are excited by stimuli contacting the tentacles, lips, and peri-oral zone, and firing of certain individual CBIs drives one or another rhythmic buccal motor program (BMP) or
components of the various interrelated programs that underlie ingestive
and egestive consummatory feeding-behaviors. Previous studies
(Teyke et al. 1990
) suggested that C-PR may affect the firing of a CBI, indicating that there is cross-communication between
the neurons involved in appetitive behaviors and those involved in
consummatory behaviors. In the present research, we examined the nature
of the synaptic input that C-PR evokes in various CBIs. The research
explored the question of whether C-PR excites all CBIs involved in
feeding, as might be expected for a neuron involved in producing a
broad undifferentiated arousal state. Alternatively, if C-PR has
differential effects on various CBIs, what are the various effects?
Could the firing of C-PR, which is involved in appetitive arousal,
contribute to consummatory behaviors and promote a subset of the
various buccal motor programs that can be generated? In answering these
questions, we also hoped to obtain evidence that could prove useful in
understanding the various roles of the very diverse population of
cerebral-buccal interneurons and more generally gain insights into the
question of how a small population of neurons controls a complex
behavior. In the course of these experiments, we identified a new
myomodulin-containing CBI that is excited by C-PR, and the present
paper describes some of the characteristics of this cell.
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METHODS |
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The experimental subjects in this research were wild-type
Aplysia californica weighing 150-400 g (Marinus, Long
Beach, CA) that were maintained and prepared for experiments as
previously described (Xin et al. 1999). More than 60 preparations were used.
Recording apparatus and bathing solutions
Intracellular recordings were obtained from isolated ganglia or from reduced preparations maintained at room temperature (17-23°C), in a clear Lucite recording chamber that was divided into two compartments, each containing artificial sea water (ASW). The first compartment contained the cerebral and buccal ganglia, whereas the second compartment contained the pedal and the pleural ganglia. The chamber contained grooves in which the C-PL and C-P connectives were placed, and the grooves were filled with petroleum jelly (Vaseline) to maintain a watertight seal between compartments. Suction electrodes were attached to selected nerves or connectives as needed for extracellular recording of unit activity. In some experiments, a solution containing increased concentrations of divalent cations (Hi-Di) was used and contained three times the normal concentration of magnesium and calcium.
Electrophysiology
For intracellular recording and stimulation, neurons were
impaled with double-barreled microelectrodes that were made of
thin-walled glass and contained 1.9 M potassium acetate and 0.1 M
potassium chloride. The electrodes were beveled so that their
impedances ranged from 10 to 15 M. For the purposes of identifying
cells and examining their morphology, the potassium acetate in the
stimulating electrode was replaced by a solution of 3%
5(6)-carboxyfluorescein dye (Kodak) in 0.05 M potassium citrate,
titrated to pH 8.0 with KOH (Rao et al. 1986
). These
electrodes were beveled so that the impedance of the electrode
containing the dye was ~10 M
and the impedance of the potassium
acetate electrode was ~6 M
. Up to three simultaneous intracellular
recordings were obtained using conventional electrometers. Nerve
recordings were made with polyethylene suction electrodes and AC
amplifiers. The nomenclature for the nerves follows that of
Gardner (1971)
.
Morphology
The locations, sizes, and shapes of cerebral neurons with
axons in the C-B connectives first were determined by back-filling the
connectives with cobalt or nickel chloride followed by treatment with
rubeanic acid (Quicke and Brace 1979). At the
termination of many electrophysiological experiments, selected neurons
were injected with carboxyfluroscein dye. To reduce the active
transport of the dye from the cells, probenecid (1 mM final
concentration) was added to the ASW bathing medium (Rosen et al.
1991
; Steinberg et al. 1987
), and the
preparation was kept for 24-48 h at 4°C. The living ganglia were
cleared in 50% glycerol in ASW and viewed with a fluorescence
microscope. Confirmation of cell morphology was made with
nickel-chloride injections followed by treatment with rubeanic acid,
fixation in paraformaldehyde, and clearing in 50% glycerol in
phosphate buffer.
Immunohistochemistry was performed on whole-mount preparations treated
as previously described (Miller et al. 1991; Xin
et al. 1999
). In some preparations, cells first were identified
and filled with biocytin. After fixation and permeabilization with Triton, the cell was labeled fluorescently by incubating the tissue with streptavidin Bodipy FL conjugate (50 µg/ml) (Molecular Probes, Eugene, OR)/PBS Triton at 4°C for 12-24 h. This then was
followed by immunostaining with a myomodulin antibody and a Cy-3 second antibody.
Identification of CBIs
A number of criteria were used to identify individual CBIs. Not all criteria were employed for every cell, but the properties of the CBIs we studied included soma location, branching patterns of the cell processes, spontaneous excitatory or inhibitory postsynaptic potentials (EPSPs or IPSPs), synaptic inputs and outputs from and to identified neurons in the buccal ganglion, and the presence of an axon in the C-B connective as determined by dye fills.
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RESULTS |
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Morphology of C-PR
Previous data indicated that stimulation of the rhinophores
or tentacles with food produces excitation of C-PR and that firing of
C-PR contributes to the evocation of a complex of responses that
represent the appetitive central motive state that precedes consummatory movements (Teyke et al. 1990). Consummatory
feeding movements are evoked when food contacts the perioral region and excites cerebral-buccal interneurons (CBIs), which drive buccal motor
programs or components of the programs. A previous study using silver
intensification of cobalt-filled cells described the basic morphology
of C-PR (Teyke et al. 1997
). Using fluorescent dye-fills, we confirmed the basic morphology of the cell, examined in
detail the location of its processes in the cerebral ganglion, and
determined the location of its cell body relative to that of the CBIs
located in the same region of the ganglion. C-PR has three main
processes that arise directly from the soma (Fig.
1A). One of the three main
trunks reaches the ventral side of the M cluster and is folded toward
the dorsal side. The second trunk of C-PR travels medially toward the C
cluster and gives off fine branches. The third trunk projects through
the cerebral-pleural connective, and previous data indicate that it
branches extensively in the ipsilateral pedal ganglion and continues to
the contralateral pedal ganglion (Teyke et al. 1997
).
The M cluster contains the somata of a number of CBIs including CBI-1,
CBI-2, CBI-3, CBI-4, and the newly identified CBI-12 (Fig.
1B). The C-PR soma is located posterior to the CBIs in the M
cluster and is just posterior to CBI-3 (Fig. 1B).
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Effects of firing of C-PR on CBI-2 and on newly identified CBI-12
Previous results suggest that the CBIs constitute a diverse
population of interneurons that may be involved differentially in the
various ingestive and egestive motor programs that the buccal ganglion
controls. We first examined the effect of C-PR on CBI-2, a CBI that
drives a robust buccal program and generates responses of the buccal
mass that resemble biting. We identified CBI-2 according to its
location in the M cluster and its ability to drive a robust BMP. Other
features that aided in the identification of the cell was the presence
of frequent multiple IPSPs and a characteristic appearance of its
processes as revealed by dye fills (Rosen et al. 1991).
In previous work, CBI-2 was shown to drive a rhythmic buccal program
(Rosen et al. 1991
). At that time, the evidence
suggested that CBI-2 was the only CBI in the M cluster region that was
capable of driving a rhythmic buccal program. In the current series of
experiments, however, it appeared that in addition to CBI-2, there was
a second cell that also could drive rhythmic buccal activity. CBI-2 and
the second cell were located close to one another at a position
adjacent to the large motor neurons (Fig. 1B) C-11 and C-12
(Rosen et al. 1991
; Teyke et al. 1993
).
The presence of a previously unidentified CBI is consistent with
nickel-chloride back-fills of the cerebral-buccal connectives (CBCs),
which indicated that the ventral M cluster may contain a CBI in
addition to the three that had been characterized previously in that
region (Rosen et al. 1991
; Xin et al.
1999
).
A preliminary screen of the two cells in the M cluster that could drive buccal programs revealed that the more laterally positioned cell was the previously reported CBI-2. This cell showed inhibition sometimes followed by some excitation when C-PR was fired (Fig. 2). For the other, more medially and superficially positioned cell, firing of C-PR produced a slow excitation (Fig. 2) that could effectively fire the cell if it was depolarized tonically or had a low resting potential (see later section). We term the more medially and superficially positioned cell CBI-12. As described in the following text, the two cells have some similarities but are different in a number of features.
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CBI-2 and CBI-12 share similar morphology
To better define the newly identified CBI-12 neuron and
distinguish it from CBI-2, we determined the effects of firing of C-PR
on the cells and then filled them with dye to determine their morphology. Nine of nine lateral cells that were presumed CBI-2 cells
were not excited by C-PR and were found to have the morphological pattern previously described for CBI-2 (Rosen et al.
1991). A dense group of processes extended directly
from the soma, primarily in the medial direction (see Fig. 3 of Rosen
et al. 1991
). This pattern is clearly different from that exhibited by
CBI-1 and CBI-3, which are located anterior and posterior,
respectively, to CBI-2. Of 11 more medially situated neurons that
received excitation from C-PR and are presumably CBI-12 cells, 6 showed
a morphology similar to that of CBI-2. In 5 of the 11 presumptive
CBI-12 neurons, however, the processes in the neuropile spread more
widely than those typically observed for CBI-2. This pattern is similar
to that exhibited by CBI-3, but the cells clearly were not CBI-3 cells,
which can be distinguished easily by their larger sizes and more
posterior positions (Fig. 1B). In three preparations, we
filled both the lateral (CBI-2) and medial (CBI-12) cells, and in all
these cases the cells could not be distinguished morphologically and
showed the CBI-2 pattern. Because the morphology of CBI-2 and CBI-12
did not clearly differentiate the cells, we studied a number of other
parameters.
CBI-2 and CBI-12 have different inputs and outputs
We examined the inputs to CBI-2 and CBI-12 from the cerebral
mechanoafferent neuron C2 and the buccal-to-cerebral interneuron B19.
These neurons previously were shown to make extensive connections to
cells in the cerebral ganglion (McCaman and Weinreich
1985; Rosen et al. 1991
; Weiss et al.
1986
). Firing of C2 produced a hyperpolarizing potential in
CBI-2 but a depolarization in CBI-12 when the two CBIs were at their
resting potential (Fig. 3A1). When both CBIs were depolarized by intracellular current to just below
threshold, C2 evoked an initial depolarization followed by a relatively
slow hyperpolarization in CBI-12. The hyperpolarization decayed back to
baseline with a time course of several seconds. By contrast CBI-2
exhibited a pure hyperpolarization that had a rapid onset and rapid
decay (Fig. 3A2).
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The two CBIs also could be distinguished from one another on the basis of the their responses to firing of neuron B19. While at resting potential, a brief burst of spikes in B19 evoked what appeared to be very similar discrete IPSPs in both CBIs (Fig. 3B1). The two CBIs behaved very differently during more prolonged firing in B19. In response to a relatively prolonged and high frequency of B19 firing, CBI-12 exhibited a barely detectable hyperpolarization followed by a substantial long-lasting depolarization that fired the cell (Fig. 3B2). By contrast, during the firing of B19, CBI-2 exhibited a weak, slow, hyperpolarization followed by a slow depolarization. The differences in input from C2 and B19 were found to be very consistent between preparations.
Not all interneurons had differential effects on CBI-2 and CBI-12. For example, buccal cerebral neuron B18 produced a slow excitation in both CBIs (Fig. 3C1) that fired the cells similarly when they were slightly depolarized (Fig. 3C2).
The rhythmic synaptic inputs the cells receive during buccal motor
programs also is different in the two CBIs. On the basis of
observations from semi-intact ganglion-buccal mass preparations (Rosen et al. 1988), during the phase of radula
retraction, a barrage of IPSPs always occurs in CBI-2. A similar
barrage of IPSPs occurs during programs elicited from isolated ganglia.
Typically after a warm-up period of one to five cycles, the IPSPs
completely block CBI-2 from firing (Fig.
4A) (see also Rosen et
al. 1991
), but the IPSPs rarely effectively block CBI-12 from
firing (Fig. 4, B and C), although they can
reduce the firing frequency of the cell.
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In addition to examining the synaptic input to the cells, we compared their ability to drive BMPs. In 41 of 43 preparations CBI-2 drove rhythmic buccal-ganglion bursts throughout a 2- to 3-min period when the cell was fired, generating 6-10 cycles of activity. By contrast, in 17 of 30 preparations, firing of CBI-12 failed to sustain rhythmic bursting and only generated two or three cycles during the 2-3 min period of stimulation. In nine preparations, both CBI-2 and CBI-12 were impaled, and in five of these preparations, both cells evoked multiple cycles of activity throughout the period that the cells were fired (Fig. 4). The bursting, however, produced by CBI-12 (Fig. 4B) was not as robust or as regular as that produced by CBI-2 (Fig. 4A), and unlike what is seen during behavioral programs, the activity in the radula nerve and nerve 2 was not well coordinated. These findings are consistent with the idea that an individual CBI-2 can initiate a functional buccal motor program, whereas CBI-12 may operate in a more modulatory role, together with other CBIs.
CBI-12 but not CBI-2 exhibits myomodulin immunoreactivity
An unidentified small cell medial to CBI-2 has been reported to be
myomodulin positive (Miller et al. 1991) and
immunocytochemistry combined with back-fills of cerebral-buccal
connectives have shown that a number of CBIs, including one in the M
cluster, are myomodulin immunopositive (Xin et al.
1999
). We therefore examined the possible myomodulin
immunoreactivity of CBI-2 and CBI-12. Myomodulin immunoreactivity was
observed in one large cell in the M cluster, identified as C12 (not
shown), and in only one other small cell the position of which
suggested that it is CBI-12. On the basis of identification by means of
electrophysiological characteristics, 11 CBI-2 and 11 CBI-12 neurons
were injected with biocytin, and the ganglia then were double stained
for myomodulin and biocytin (Fig.
5A). The results indicated
that all of the CBI-12 cells were myomodulin positive, whereas none of
the CBI-2 cells were myomodulin positive (Fig. 5B).
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Firing of C-PR evokes voltage-dependent excitation of CBI-12
The excitatory effect of C-PR on CBI-12 varied significantly between preparations: in some preparations a strong excitation of >15 mV was recorded, whereas in other preparations the depolarization was much smaller. Variations of the magnitude appeared to be related to the membrane potential of the cell. Indeed, we found that when CBI-12 was undamaged, and at its resting potential, firing of C-PR produced relatively little and sometimes no obvious depolarization of the cell. A clear excitation was observed, however, when CBI-12 was depolarized 5 mV by means of constant current injection (Fig. 6A). When CBI-12 was predepolarized by >10 mV, C-PR firing brought CBI-12 above firing threshold (Fig. 6B). The EPSP evoked in a tonically depolarized cell, greatly outlasted the duration of the firing of C-PR, but the expression of the slow EPSP could be terminated immediately by stepping the cell membrane potential back to rest (Fig. 6C, 1st set of traces). Conversely if C-PR was fired while CBI-12 was at resting potential (Fig. 6C, 2nd set of traces), no depolarization was evident in CBI-12, but a slow depolarization could be uncovered, by passing a constant depolarizing current into the cell at the termination of firing of C-PR (n = 4). The overall data suggest that firing of C-PR produces a long-duration voltage-dependent EPSP in CBI-12.
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Effect of C-PR on CBIs is polysynaptic
Previous studies (Teyke et al. 1997) provided
evidence that the effects of C-PR on cerebral B motor neurons and on
the metacerebral cell are mediated by interneurons located in the pedal
or pleural ganglion. To determine the location of possible interneurons
mediating the effects of C-PR on CBI-12, we placed the cerebral
ganglion and the pedal-pleural ganglia in different chambers, leaving
the connectives intact. Thus we could change selectively the solution bathing the cerebral or pedal-pleural ganglia. We studied the effect of
raising the firing threshold of pleural and pedal-ganglion neurons by
first establishing that firing of C-PR evoked a depolarization in
CBI-12 and then determining if the effect remained after bathing the
pedal-pleural ganglia in a solution containing increased divalent cations (Hi-Di, 3 times normal Ca; 3 times normal Mg)
(n = 4). For conditions shown in Fig.
7, C-PR was fired similarly as indicated by the bottom recording. In these experiments, we also
recorded extracellularly from a CBC to monitor the activity of other
CBIs the firing of which was affected by C-PR. In normal ASW, firing of
CBI-12 depolarized CBI-12 and also excited other CBIs as indicated by
the recording from the CBC (Fig. 7A). Exposure of the
pedal-pleural ganglia to Hi-Di completely blocked the effect of firing
of C-PR on CBI-12 and on other CBIs recorded in the CBC (Fig.
7B). Partial recovery of the responses occurred when the
pedal-pleural ganglia were re-exposed to ASW (Fig. 7C).
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Effects C-PR firing are mediated by ipsilateral and contralateral cerebral-pedal connectives
It was shown previously that the effect of firing C-PR on cerebral
neurons such as the MCC and B cells was mediated by pedal-pleural interneurons that send their axons through cerebral-pedal or
cerebral-pleural connectives (Teyke et al. 1997). To
explore the pathway by which C-PR affects CBIs, ipsilateral or
contralateral pedal or pleural connectives were sequentially sectioned
(n = 4). The effect of firing of C-PR on the
excitability of CBI-12 or the activity of other CBIs recorded from a
CBC was not substantially altered by cutting both cerebral-pleural
connectives (Fig. 8, A and
B). Cutting a single cerebral-pedal connective contralateral
to C-PR reduced the effects of C-PR on CBIs (Fig. 8C),
whereas cutting both pedal-cerebral connectives completely blocked the
effects of C-PR (Fig. 8D). Similar results were seen in
those preparations in which C-PR had some actions on CBI-3
(n = 3). The order in which the connectives were cut
did not appear to be significant as long as the last connective cut was
the ipsilateral pedal-cerebral connective (which contains the C-PR axon
that projects to the pedal-pleural ganglion).
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Effects of pleural and pedal ganglion neurons on CBIs also may involve cerebral interneurons
Although the effects of firing of C-PR on CBI-12 and other CBIs appear to be mediated by interneurons located in the pedal-pleural ganglia, it is possible that these interneurons do not directly innervate the CBIs but rather synapse on intermediate interneurons located in the cerebral ganglion. To test this possibility, the cerebral ganglia but not the pedal-pleural ganglia were bathed in a Hi-Di solution while C-PR was fired. Under these conditions, the effect of firing of the C-PR also was blocked (n = 4) (normal ASW, Fig. 9, A1 vs. high-divalent sea water, A2). In two experiments on CBI-3, in normal ASW firing of C-PR evoked what appeared to be a mixture of excitatory and inhibitory synaptic potentials (Fig. 9B1), and these effects were blocked (Fig. 9B2) when the concentration of calcium in the solution was increased to five times normal. The high-calcium solution raises cell thresholds while presumably not decreasing synaptic output of neurons. In these experiments, the effects of the divalent cations were not clearly reversed when the preparation was returned to ASW.
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Different CBIs exhibit various inputs when C-PR is fired
Twelve CBIs have been identified, and most of them have different
but characteristic effects on buccal programs (Perrins and Weiss
1998; Rosen et al. 1991
; Xin et al.
1999
), suggesting that different CBIs or different combinations
of CBIs may evoke or modulate the various behaviors that are known to
be mediated by the buccal ganglion. We therefore examined the effect of
firing of C-PR on various CBIs, concentrating on those that can be
approached from the ventral surface of the cerebral ganglion (the
surface containing the cell body of C-PR).
Firing of C-PR evoked a burst of what appeared to be discrete polysynaptic EPSPs in CBI-1 (Fig. 10A). Firing of C-PR also evoked a burst of PSPs in CBI-3, but the PSPs were relatively small and appeared to be comprised of both EPSPs and IPSPs (Fig. 10B). As shown in a later section, based on changes in the excitability of CBI-3 the cell apparently receives waves of inhibitory inputs when C-PR is fired (see Fig. 14). CBI-4 received little or no input when C-PR was fired.
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Effect of C-PR on plateau potentials of CBI-5/6
Firing of C-PR did not appear to evoke any obvious synaptic input
into the newly identified pair of CBIs, CBI-5/6 (Perrins and
Weiss 1998), either at resting potential or when CBI-5/6 was depolarized. Furthermore, firing of C-PR did not produce any change in
the input resistance of CBI-5/6, as measured with constant-current hyperpolarizing pulses (Fig.
11A).
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Because CBI-5/6 can generate plateau potentials (Perrins and
Weiss 1998), we determined the effect of firing of C-PR on the capacity of CBI-5/6 to initiate these responses. The amount of current
needed to produce a reliable plateau response first was determined for
a given CBI-5/6 (Fig. 11B), and that level then was
presented repeatedly (note that under conditions in which repeated
pulses are given, the plateau response of CBI-5/6 appears as an active
response that does not outlast the depolarizing pulse). When a 6-s
burst of spikes was evoked in C-PR, the capacity of CBI-5/6 to generate
a plateau potential was suppressed even though no obvious IPSP could be
observed (Fig. 11C; n = 4). The onset of the
suppression of the plateau was typically very slow and often not
clearly present until the 6-s burst of C-PR spikes was terminated.
Effects of C-PR on CBI 8/9
Simultaneous recordings from dorsal CBIs that have been identified
recently (Xin et al. 1999) and the ventrally located
C-PR were obtained by pinning the ganglion, dorsal side up, and
twisting the anterior portion of the cerebral ganglion so that C-PR was exposed. CBI-8 and -9 consist of two similar cells located at the base
of the AT nerve. These cells received a slow excitation when C-PR was
fired (Fig. 12).
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Effects of C-PR on the excitability of CBIs
Although C-PR excited a number of CBIs, activity of C-PR never
evoked buccal motor programs even when both C-PRs were fired at high
frequency. Furthermore we have found that firing of C-PR leads to an
inhibition in many buccal CPG elements such as B31/B32, B63, and B34
(Hurwitz, Weiss, and Kupfermann, unpublished observations). Therefore
the actions of C-PR may be primarily modulatory and might be most
apparent when it is active together with other inputs to the CBIs. To
reveal possible modulatory effects of C-PR, CBIs were injected
suprathreshold constant current depolarizing pulses that were adjusted
so that they evoked three to five action potentials. We then determined
whether the number of evoked spikes was affected by C-PR, which was
fired by constant current pulses adjusted to evoke a 6-s train of
spikes at ~20 Hz, which is within a physiological range (Teyke
et al. 1991).
The effect of firing of C-PR was determined for four to eight cells of a given type in three to six runs. C-PR primarily enhanced the excitability of CBI-1, CBI-8/9, and CBI-12 (Fig. 13, A-C) and had no effect on the excitability of CBI-4 (Fig. 13D). The effect of C-PR on CBI-2 consisted of a very small decrease of excitability (Fig. 13E). CBI-3 exhibited multiple effects (Fig. 13F): an early decrease of excitability followed by an increase of excitability and finally a second decrease of excitability.
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We obtained more quantitative data on CBI-1, -2, -3, -4, -8/9, and -12 by recording from six identical cells for at least three runs for each cell, and averaging the number of action potentials per cell per run (3 times before C-PR was fired, 3 times while C-PR was fired and 11 times after termination of C-PR firing). The data were plotted as number of evoked spikes as a percent of initial control. Figure 13 shows the average and standard error of six normalized individual runs for each of the six CBIs that was tested, before, during, and after C-PR was fired (period of firing indicated by thick horizontal bars). The data confirm our previous observations. CBI-12 exhibited a large and relatively prolonged increase in excitability. CBI-1 exhibited a smaller increase of excitability, CBI-8/9 exhibited a yet smaller increase of excitability, and CBI-4 exhibited only a very small increase in excitability. CBI-2 appeared to exhibit a small decrease of excitability, and finally CBI-3 showed an initial decrease followed by a return to baseline or small increase of excitability and finally a late inhibition of long duration. Note that in Fig. 13 the vertical scales for the effects on CBI-3, -2, and -4 have been magnified compared with the scales for CBI-12, -1, and -8/9.
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DISCUSSION |
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These studies illuminate two questions. Does a neuron such as C-PR, which plays a role in generating a motivational state, interact directly or indirectly with command-like neurons for specific behaviors? Can a population of command-like neurons be differentiated on the basis of synaptic input from C-PR?
The cerebral ganglion contains a small number of neurons that
project axons out the cerebral-buccal connective. Some of these are the
somata of primary mechanoafferents, and one is the extrinsic modulatory
neuron, the metacerebral cell. The remaining neurons are termed CBIs
and have been postulated to be involved in the generation of one or
more of the stereotyped motor programs generated by the buccal
ganglion. The CBIs consist of ~12 cells. The buccal ganglion
generates what appear to be at least six distinct but related motor
programs (grazing, rejection, withdrawal, biting, swallowing, and
cutting) (Howells 1942; Hurwitz and Susswein
1992
; Kupfermann 1974a
; Kupfermann and
Carew 1974
; Morton and Chiel 1993
), and thus it
is likely that at least some of these behaviors involve the combined
activity of several CBIs. Consistent with this suggestion are the
observations that buccal interneurons and afferent stimulation, provide
synaptic inputs to more than one CBI at a time (Rosen et al.
1991
). One approach to understanding how the various CBIs act
together to generate behavior is to examine their differential inputs.
In the current study, we determined the effects of firing of C-PR on
CBIs. C-PR was of particular interest because there is considerable
evidence that it is an important element of the neural circuitry that
generates various manifestation of the appetitive arousal that precedes
some consummatory behaviors. For example, the cell fires when animals
assume the head-up feeding posture (Teyke et al. 1991
),
and animals exhibit deficits in the intensity and rate of consummatory
feeding responses when the connectives that contain the C-PR axon are
severed (Kupfermann 1974b
).
Cerebral M cluster contains a novel myomodulin-containing CBI
We examined the input to a variety of identified CBIs located in
the M and E clusters of the cerebral ganglion. In the course of these
studies, we encountered a previously undescribed CBI, which we have
named CBI-12. Previous studies reported that only a single cell in the
M cluster drives a maintained buccal motor rhythm. In the current
research, we found a second M cluster neuron (termed CBI-12) that can
drive rhythmic activity, although compared with CBI-2, the rhythm
driven by CBI-12 is not as robust or coordinated between various buccal
nerves. Thus the effects of CBI-12 may be primarily in modulating an
ongoing program rather than in initiating a complete program. The
morphology of CBI-12 was similar to that of CBI-2, and it is possible
that previous studies of CBI-2 on occasion were performed on CBI-12. We
now find, however, that the two cells can be distinguished based on a
combination of features, including their exact position in the M
cluster, the nature of the programs they evoke, and inputs from sensory
cells (such as C2) and interneurons (such as B19 and C-PR). The single
feature that most definitively distinguishes the cells is that CBI-12 exhibits immunoreactivity to the peptide, myomodulin (MM), whereas CBI-2 is myomodulin negative. The MM immunoreactivity of CBI-12 is
consistent with the previous evidence that the cerebral ganglion transports large amounts of MM to the buccal ganglion (Lloyd
1988).
C-PR, a neuron involved in appetitive arousal, has differential effects on cerebral-buccal interneurons
Firing of C-PR was found to evoke strong synaptic input to some CBIs but little or no input to other CBIs. Furthermore for the CBIs that received input, some were excited, some inhibited, and one pair (CBI-5/6) appeared to exhibit a change in an intrinsic membrane property that affected their capacity to exhibit plateau potentials. The synaptic effects of C-PR on CBIs are not consistent with the hypothesis that C-PR produces an indiscriminate general arousal but rather are most compatible with the idea that the firing of C-PR promotes the activity of only a subclass of feeding-related behaviors, such as grazing during locomotion, rather than biting, during stationary feeding (see next section).
Although both CBI-12 and CBI-2 can drive rhythmic buccal ganglion
activity, the two cells receive very different synaptic inputs when
C-PR is fired. C-PR primarily inhibits CBI-2. By contrast, C-PR evokes
a slow EPSP in CBI-12. The effect of the EPSP is modulatory in that the
amplitude of the EPSP is enhanced greatly when the cell is depolarized.
In other words, C-PR on its own will not have much effect on CBI-12 but
will modulate the effects of inputs that depolarize CBI-12. Other cells
that are excited by firing of C-PR are CBI-8/9, a pair of cells that
are similar to CBI-12 in that they contain myomodulin. Similar to
CBI-12, CBI-8/9 can drive rhythmic buccal activity, but the rhythm is
not robust and appears to include primarily neurons in buccal nerve 3 rather than in other buccal nerves (Xin et al. 1999).
CBI-3, a cell that appears to inhibit ongoing buccal motor programs
(Rosen et al. 1991
), receives a slow inhibition when
C-PR is fired, whereas CBI-1, which drives a single cycle of a buccal
motor program, receives relatively weak excitation. It may be
significant that CBI-2 and CBI-4, the two cells in which C-PR produces
the least modulation of their firing, drive the most robust and
reliable rhythmic buccal motor programs. Thus it is possible that the
cells that receive more substantial inputs from C-PR and from the
interneurons that C-PR drives function more in a modulatory role in
adjusting ongoing programs rather than directly eliciting programs.
C-PR may promote a subset of feeding-related motor programs
Our data suggest that the synaptic inputs evoked by firing C-PR are mediated by interneurons that are located in the pedal or pleural ganglia and that send their axons to the cerebral ganglion via the cerebral-pedal connectives and possibly excite secondary cerebral interneurons. Most of the effects of C-PR on the CBIs appear to involve slow onsets and decay, because they either are mediated by slow synaptic potentials or are mediated by small fast potentials produced by interneurons that have slow onset and offset times. In either case, the effects of C-PR do not appear to be appropriate for producing phasic effects but rather appear to involve relatively slow changes in the excitability of the CBIs.
Because one of the main effects of C-PR on the pedal ganglion is to
excite neurons that are involved in generating a head-up posture, it is
possible that the interneurons that mediate the effect of C-PR on the
CBIs are part of the neural circuitry involved in the head-up posture
or other aspects of appetitive arousal. Thus it can be postulated that
those CBIs that are excited by these interneurons may be active during
the ingestive behaviors that are associated with appetitive arousal.
Neurons that are inhibited or receive no input may be related to
egestive behaviors. Alternatively these CBIs may be related to
ingestive behaviors, such as grazing (Kupfermann and Carew
1974), that occur in the absence of a head-up posture. Previous
data suggested that firing of C-PR excited pedal or pleural
interneurons that mediate synaptic excitation of the serotonergic
metacerebral cells or inhibition of Bn cells, which appear to be
involved in defensive withdrawal responses (Teyke et al.
1989
). The inputs to the Bn cells appeared to travel via the
cerebral-pleural connectives, whereas the inputs to the MCC travel via
the cerebral-pedal connectives. Thus the inhibitory input to the Bn
cells must be mediated by a set of interneurons that are different from
those that excite the MCC and the CBIs (Teyke et al.
1997
).
It was suggested previously that one reason the connections of C-PR to
cerebral neurons such as Bn cells and the MCC take an indirect route,
via the pedal ganglion, is that this serves to ensure appropriate
sequencing of feeding behavior (Teyke et al. 1997).
Feeding involves initial appetitive responses that are mediated by
neurons located in the pedal and pleural ganglia. These responses
precede consummatory responses, which are mediated by neurons contained
primarily in the cerebral and buccal ganglia. The current data suggest
that food stimuli initially may activate C-PR, which then activates
pedal ganglion circuitry, which in turn activates a specific subset of
CBIs. Thereby the appetitive aspects of feeding (head lifting and
orientation) will be promoted to precede specific classes of the
consummatory aspects of feeding. It is of interest that based on the
use of high-divalent cation solutions, our evidence suggests that at
least part of the effect of the presumptive pedal-pleural interneurons
that mediate the effects of C-PR on CBIs may involve intermediary
interneurons located in the cerebral ganglion.
The differential input to CBIs cells from C-PR suggests that one form
of feeding behavior may be potentiated by circuitry associated with
generating a head-up posture, whereas another form of feeding may be
depressed by the head-up circuitry. Behavioral observations indeed
suggest that feeding may occur either during the head-up posture or
during grazing, during which the head of the animal is on the substrate
and the animal slowly locomotes while feeding (Kupfermann and
Carew 1974). A testable prediction of this hypothesis is that
circuitry associated with locomotion may have actions opposite to those
produced by firing of C-PR, namely excitation of CBI-2 and inhibition
of CBI-12. Definite conclusions about the specific role of C-PR in the
generation or modulation of buccal programs must await further
information about the firing patterns and rate of C-PR and of the CBIs
during the various behaviors that are mediated by the buccal ganglion.
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ACKNOWLEDGMENTS |
---|
We thank A. Klein and S. Rosen for comments on the study.
This work was supported by National Institutes of Health Grants MH-50235, MH-36730, GM-320099, and K05-MH-01427 and by Human Frontier Science Program LT-0464/1997.
Present address of R. Perrins: School of Biological Sciences, Woodland Road, University of Bristol, Bristol BS8 1UG, UK.
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FOOTNOTES |
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Address for reprint requests: I. Kupfermann, Center for Neurobiology and Behavior, Columbia University, 722 W. 168 St., Box 25, New York City, NY 10032.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 6 March 1998; accepted in final form 22 October 1998.
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REFERENCES |
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