1Department of Neurobiology and Anatomy and W. M. Keck Center for the Neurobiology of Learning and Memory, The University of Texas-Houston Medical School, Houston, Texas 77225; and 2N. K. Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow 117808, Russia
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ABSTRACT |
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Kabotyanski, Evgeni A., Douglas A. Baxter, Susan J. Cushman, and John H. Byrne. Modulation of Fictive Feeding by Dopamine and Serotonin in Aplysia. J. Neurophysiol. 83: 374-392, 2000. The buccal ganglia of Aplysia contain a central pattern generator (CPG) that mediates rhythmic movements of the buccal apparatus during feeding. Activity in this CPG is believed to be regulated, in part, by extrinsic serotonergic inputs and by an intrinsic and extrinsic system of putative dopaminergic cells. The present study investigated the roles of dopamine (DA) and serotonin (5-HT) in regulating feeding movements of the buccal apparatus and properties of the underlying neural circuitry. Perfusing a semi-intact head preparation with DA (50 µM) or the metabolic precursor of catecholamines (L-3-4-dihydroxyphenylalanine, DOPA, 250 µM) induced feeding-like movements of the jaws and radula/odontophore. These DA-induced movements were similar to bites in intact animals. Perfusing with 5-HT (5 µM) also induced feeding-like movements, but the 5-HT-induced movements were similar to swallows. In preparations of isolated buccal ganglia, buccal motor programs (BMPs) that represented at least two different aspects of fictive feeding (i.e., ingestion and rejection) could be recorded. Bath application of DA (50 µM) increased the frequency of BMPs, in part, by increasing the number of ingestion-like BMPs. Bath application of 5-HT (5 µM) did not significantly increase the frequency of BMPs nor did it significantly increase the proportion of ingestion-like BMPs being expressed. Many of the cells and synaptic connections within the CPG appeared to be modulated by DA or 5-HT. For example, bath application of DA decreased the excitability of cells B4/5 and B34, which in turn may have contributed to the DA-induced increase in ingestion-like BMPs. In summary, bite-like movements were induced by DA in the semi-intact preparation, and neural correlates of these DA-induced effects were manifest as an increase in ingestion-like BMPs in the isolated ganglia. Swallow-like movements were induced by 5-HT in the semi-intact preparation. Neural correlates of these 5-HT-induced effects were not evident in isolated buccal ganglia, however.
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INTRODUCTION |
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The feeding behavior of Aplysia
consists of a sequence of appetitive and consummatory behaviors
(Kupfermann 1974a,b
; Kupfermann et al.
1991
). First, food (seaweed) in the immediate environment of
the animal activates appetitive behaviors, such as locomotion and head
waving, which bring the animal in contact with the food (Teyke
et al. 1990
, 1992
). Second, contact with the food activates consummatory behaviors, such as ingestion (biting and swallowing) or
rejection (Kupfermann 1974a
; Susswein et al.
1976
). Consummatory behaviors involve rhythmic movements of
feeding organs, including the lips, jaws, buccal mass, odontophore, and
esophagus (Drushel et al. 1997
; Kupfermann
1974a
; Morton and Chiel 1993a
; Rosen et al. 1997
; Weiss et al. 1986
). During a bite, the
odontophore is rotated forward (i.e., protraction) as the jaws open.
Initially, the two halves of the radula (toothed grasping surfaces of
the odontophore) are separated during protraction. Before the peak of
protraction, however, the radula begins to close and grasp the food.
The radula remains closed as the odontophore retracts (backward
rotation), which brings the food into the buccal cavity, and the jaws
close. Swallowing consists of rhythmic movements of the odontophore and
radula, which are similar to those during biting, and is associated
with peristaltic contractions of the esophagus. Unlike biting,
swallowing is not associated with opening of the jaws. In addition to
biting and swallowing, the feeding apparatus can produce rejection
movements in response to inedible material in the buccal cavity. During
rejection, the radula is closed as the odontophore protracts and open
as it retracts, causing the unwanted material to be ejected from the
buccal cavity. Thus the motor programs that mediate consummatory
feeding behaviors can be described, in general, as having two phases: a
protraction phase followed by a retraction phase. During ingestion, the
radula is open during the initial phase of protraction and closed
during retraction, whereas during rejection, the radula is closed
during protraction and open during retraction. Thus the two-phase
feeding motor program of Aplysia, which is a browser,
differs from other herbivorous gastropods (e.g., Helisoma, Helix,
Limax, Lymnaea, Planorbarius, etc.), which are raspers and have
three phases of feeding cycle (for review, see Kupfermann
1974a
,b
; Willows 1985
).
Using a variety of intact, semi-intact, and reduced preparations,
recent studies have begun to relate specific patterns of neural
activity recorded in vivo and in situ to aspects of consummatory feeding behaviors (Chiel et al. 1986; Church and
Lloyd 1994
; Cropper et al. 1990
; Evans
and Cropper 1997
; Evans et al. 1996
;
Fiore et al. 1992
; Hurwitz et al. 1996
;
Jahan-Parwar and Fredman 1983
; Kabotyanski et al.
1997
, 1998a
; Kupfermann and Weiss 1982
;
Morton and Chiel 1993a
,b
; Nagahama and Takata
1987
, 1988
, 1990
; Nargeot et al. 1997
, 1999a
-c
;
Perrins and Weiss 1996
, 1998
; Rosen et al. 1991
,
1997
; Scott et al. 1995
; Susswein et al.
1996
; Weiss et al. 1978
, 1986
). Two
types of buccal motor programs (BMPs) have been characterized during
recordings in vivo. One type of BMP was associated primarily with
ingestion, whereas the other was associated primarily with rejection
(Morton and Chiel 1993a
). The two BMPs were
distinguished by the timing of large-unit activity in the radula nerve
(R n.) relative to the onset of large-unit activity in buccal nerve 2 (n.2). During ingestion, large-unit activity in the two nerves
overlapped to a large degree. In contrast, during rejection, large-unit
closer activity in R n. preceded large-unit activity in n.2. Moreover,
some motor neurons that contribute to the extracellular recordings have
been identified (e.g., Hurwitz et al. 1996
;
Morton and Chiel 1993b
; Nargeot et al.
1997
). Some of the large units that were recorded in the R n.
corresponded to activity in radula-closer motor neuron B8, whereas some
of the large units recorded in the n.2 corresponded to activity in
radula-retractor motor neurons B10 and B9. Thus the timing of activity
in radula-closure motor neurons relative to activity in
radula-retractor motor neurons was predictive of the type of
consummatory behavior (i.e., ingestion or rejection) in the intact animal.
The in vivo studies have provided a framework, or set of criteria, that
can be used to evaluate the potential behavioral relevance of BMPs
observed in more reduced preparations and/or in response to different
stimuli or experimental conditions. Using these criteria, several
studies have found that the isolated buccal ganglia retain the
circuitry necessary to produce at least two behaviorally relevant patterns (i.e., rejection-like and ingestion-like BMPs) and that the
central pattern generator (CPG) in the buccal ganglia can switch
between different functional configurations (e.g., Hurwitz and
Susswein 1996; Hurwitz et al. 1997
;
Kabotyanski et al. 1997
, 1998a
; Morton et al.
1991
; Nargeot et al. 1997
, 1999a
,b
;
Plummer and Kirk 1990
; Rose 1972
;
Sossin et al. 1987
; Susswein and Byrne 1988
). The mechanisms underlying the generation of the
different BMPs and the switching between different BMPs is not well
understood, however.
Several lines of evidence suggest that the generation and switching
among different BMPs may be mediated, in part, by the actions of
catecholamines (e.g., dopamine, DA) and/or the indolamine serotonin
(5-HT). First, the CPG of the buccal ganglia contains an intrinsic
system of putative DA-containing cells (e.g., cells B20 and B65)
(Kabotyanski et al. 1994, 1998a
; Teyke et al.
1993
) and receives extrinsic catecholaminergic inputs (e.g.,
cell CBI-1) (Rosen et al. 1991
). Second, direct
depolarization of these cells elicits rhythmic neural activity in the
CPG and, in some instances, may selectively elicit ingestion-like BMPs.
Third, the CPG of the buccal ganglia receives extrinsic serotonergic
inputs (e.g., the serotonergic metacerebral cell, MCC) (Weiss et
al. 1978
, 1981
; see also Goldstein and Schwartz
1989
; Rathouz and Kirk 1988
; Salimova et
al. 1987
; Soinila and Mpitsos 1991
;
Susswein et al. 1993
). Fourth, firing MCC increases the
rate of BMPs under some experimental conditions (Kupfermann et
al. 1979
; Weiss et al. 1978
). Finally, in vivo
recordings from freely behaving animals indicate that MCC is active
during feeding (Fiore et al. 1992
; Kupfermann and Weiss 1982
; Weiss et al. 1978
) and that lesions
of the MCC induced deficits in biting (Rosen et al. 1983
,
1989
). These results suggest that DA and 5-HT may play roles in
initiating rhythmic activity in the CPG and, more specifically, in
organizing those BMPs that underlie aspects of ingestion.
To further characterize the roles of DA and 5-HT in the feeding system
of Aplysia, the present study examined the effects of DA
and 5-HT in progressively more reduced preparations. Perfusing a
semi-intact head preparation with DA (50 µM) induced feeding-like movements that were similar to bites in the intact animal. Similarly, perfusing isolated ganglia preparations with DA (50 µM) elicited ingestion-like BMPs. Serotonin (5 µM) also induced feeding-like movements when perfused into the semi-intact preparation. The 5-HT-induced movements were similar to swallows, however. Perfusing isolated buccal ganglia with 5-HT (5 µM) did not significantly change
the number of BMPs nor did it significantly bias the output of the CPG
toward ingestion-like BMPs. Finally, the effects of DA and 5-HT on
several cells (e.g., B4/5, B8, B31/32, B34, B35, B51, B63, and B64) and
synaptic connections within the CPG were examined. The results of the
present study suggest that DA and 5-HT regulate the functional
configuration of the CPG and thereby play distinctive roles in
organizing different aspects of feeding. Preliminary reports of some of
these results have appeared in abstract form (Baxter and Byrne
1993; Baxter et al. 1995
; Cushman et al.
1995
; Kabotyanski et al. 1993
, 1995
, 1998b
).
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METHODS |
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Aplysia californica (150-300 g) were obtained from Alacrity Marine Biological Services (Redondo Beach, CA), Marine Specimens Unlimited (Pacific Palisades, CA), Marinus (Long Beach, CA), and Pacific Biomarine (Venice, CA). Animals were kept in aerated aquaria containing artificial seawater (ASW; Instant Ocean, Aquarium Systems, Mentor, OH), which was maintained at 15°C. Animals were fed dried seaweed (Hang Loong Marine Products, Japan) three times a week. Before dissections, animals were anesthetized by an injection of isotonic MgCl2.
Semi-intact head preparation
The semi-intact head preparation (Fig.
1) consisted of the anterior portion of
the animal (i.e., an isolated head) that retained, in situ, the
external structures of the head (e.g., lips, tentacles, and
rhinophores), the feeding organs (e.g., jaws, buccal mass, and
esophagus), and the buccal, cerebral, pleural, and pedal ganglia (see
also Chiel et al. 1986; Drushel et al.
1997
; Nagahama and Takata 1987
, 1988
;
Rosen et al. 1991
; Weiss et al. 1986
).
Before each experiment, animals were food-deprived for 2 days. The head was cut from the rest of the animal slightly rostral to the parapodia (dorsally) and slightly caudal to the beginning of the foot
(ventrally). The esophagus was severed rostral to the crop and was
canulated with a polyvinyl tube, 80 mm long and 3 mm in diameter. The
anterior aorta was severed slightly caudal to the pedal artery. Another polyvinyl tube, 80 mm long and 2 mm in diameter, was inserted in the
aorta and protruded rostrally into the buccal artery. One ligature
secured the buccal artery to the tube, and another constricted the
anterior aorta so that the perfusion of the buccal artery bypassed the
pedal and cephalic arteries. The peripheral nerves from the
pleural-pedal ganglia that projected caudally (e.g., PL2,
pleuroabdominal connectives, P7, P8, P9, and P10) were severed. Once
the gross dissection was completed, the head was transferred to an
experimental perplex chamber, which contained ASW. Within the chamber,
the preparation was mounted on a polyurethane tube, which had a
diameter similar to that of the "neck" of the isolated head. The
head was mounted on the tube by pinning the cut edges of the neck to
the tube as well as by tying a ligature around the overlapping portions
of the neck and tube. The polyurethane tube was attached on a perplex
tube that was attached to one wall of the experimental chamber. This
wall had sliding gate/door (not shown) through which the two canula
were drawn outside the chamber. The gate then was closed and sealed off
with petroleum jelly (Vaseline). Thus the preparation had two isolated
compartments: one outside the head and another inside (Fig. 1). The
volume of the internal compartment (30-40 ml) depended on the size of
the head.
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The canulated buccal artery was used to pump ASW into the arterial system and thereby perfuse the tissue and simulate the normal hydroskeletal inflation of the animal. The rate of perfusion was 1-2 ml/min. To maintain hydroskeletal inflation, positive hydrostatic pressure was provided inside the head by elevating the open end of the outflow tube 30-40 mm above the surface of ASW in the chamber. In addition, the canulated aorta was used to perfuse drugs (e.g., DA or 5-HT) into the preparation. Note that the drugs in chosen concentrations (see following text) were applied directly to the buccal ganglia, buccal mass, and the body wall. They reached the cerebral, pedal, and pleural ganglia much diluted and delayed. (If the volume of internal head compartment was 30 ml, perfusing it with a drug at 1 ml/min rate would reach half the final concentration in ~30 min)
The esophagus was canulated for two reasons: first, to prevent exchange of fluids between internal and external compartments of the head preparation through the buccal orifice and, second, to control a thread that was fed through the radula. The thread was used to measure feeding movements. It was attached to a lever of Isotonic Transducer (Model 60-3000, Harvard Apparatus, Natick, MA) and was drawn into the mouth, between the two halves of the radula and through the esophagus (Fig. 1). It was adjusted before each experiment to set the initial point of recording. A counterbalance weight was attached to the opposite arm of the transducer's lever so that a small constant outward force of 0.2 g was applied to the thread, ensuring that the thread always was stretched and transduced movements in both directions. The signal from the force transducer was amplified further with an oscilloscope, displayed on a chart recorder, and stored on magnetic tape. In addition, two video cameras and a video mixer were used to make a video tape record of the semi-intact preparation. One camera was focused on the mouth of the isolated head preparation, and the other was focused on the signal from the force transducer, which was displayed on the chart recorder. The two simultaneous video images were combined by the video mixer into one image, which was displayed and stored on video tape for subsequent analysis. Experiments were performed at 15°C.
In vitro preparation
An isolated buccal ganglia preparation was used to make
extracellular recordings of BMPs and intracellular recordings from identified cells. Typically the peripheral nerves of the buccal ganglia
were severed at the point where they entered the buccal mass,
esophagus, and salivary glands. The cerebral-buccal connectives (C-B
conn.) were severed close to the cerebral ganglion. The buccal ganglia
were removed from the animal and pinned to the floor of a recording
chamber, which was coated with a silicone elastomer (Sylgard 184, Dow
Corning, Midland, MI). In those preparations where only extracellular
recordings were made, the connective tissue sheath that covers the
buccal ganglia was left intact and the cut ends of the R n. and buccal
nerves 1, 2, and 3 (n.1, n.2, and n.3) were drawn into extracellular
electrodes. (The nerve designations are from Gardner
1971; see also Church and Lloyd 1994
;
Morton and Chiel 1993b
; Scott et al.
1991
.) In those preparations in which both intracellular and
extracellular recordings were made, the buccal ganglia were pinned to
the floor of the recording chamber so that the rostral surface of the
right buccal ganglion and caudal surface of the left buccal ganglion
were accessible. The ganglia were desheathed, and only the R n. was
drawn into an extracellular electrode. For intracellular recordings,
neurons were identified by their position, size, projection of axons in peripheral nerves, nature of synaptic connectivity, characteristic patterns of firing during BMPs, and intrinsic biophysical properties (see RESULTS for details).
Conventional techniques were used for extracellular and intracellular
recordings of neural activity. Briefly, extracellular recordings of
neural activity were made with polyethylene suction electrodes and AC
coupled amplifiers (Model 1700, AM Systems, Everett, WA). The suction
electrodes also were used to stimulate buccal nerves. Intracellular
electrodes were filled with a solution of 3 M potassium acetate and 100 mM potassium chloride and had an impedance of 6-15 M. The signals
from the intracellular electrodes were amplified using DC-coupled
amplifiers (models Axoclamp 2A and Axoprobe 1A, Axon Instruments,
Burlingame, CA). The extracellular and intracellular signals were
amplified further and displayed on a chart recorder, and an
oscilloscope and were recorded on magnetic tape.
Solutions
Generally, control saline consisted of ASW, to which 10 mM of either TRIZMA or HEPES buffer (Sigma Chemical, St. Louis, MO) was added and the pH was adjusted to 7.4. Solutions containing L-3,4-dihydroxyphenylalanine, a metabolic precursor of catecholamines (DOPA; Calbiochem, La Jolla, CA), 3-hydroxytyramine HCl (dopamine, DA; Calbiochem), and 5-hydroxytryptamine creatinine sulfate complex (serotonin, 5-HT; Sigma Chemical) were made immediately before use. Solutions of DA and DOPA also contained an equimolar concentration of an antioxidant (ascorbic acid; Sigma Chemical). In those experiments in which either DA or DOPA was used, the control saline also contained ascorbic acid.
Unless otherwise noted, 50 µM DA, 250 µM DOPA, or 5 µM 5-HT was
used. These concentrations were selected on the basis of preliminary studies that examined the effects of 1-500 µM DA, 0.5-50 µM 5-HT, and 100-1,000 µM of DOPA. Concentrations were selected that elicited reliable, stable, but not maximal responses. Moreover these
concentrations were similar to concentrations used in previous studies
(e.g., Ascher 1972; Gospe and Wilson
1980
; Kabotyanski et al. 1994
; Kramer and Levitan 1990
; Ocorr and Byrne 1985
;
Shozushima 1984
; Sossin et al. 1987
;
Teyke et al. 1993
; Weiss and Drummond
1981
; see also Gospe 1983
; Trimble and
Barker 1984
; Yeoman et al. 1994
). In previous studies, levels of DA and 5-HT were measured in the CNS, specific ganglia and their neuropils, monoamine-containing neurons, and even
vesicles (e.g., Chien et al. 1990, 1995; McCaman
et al. 1973
, 1979
). These measurements, however, do not
provide information about effective concentrations acting at synaptic
contacts. It is possible to estimate such concentrations by comparing
cellular responses to exogenous transmitters with responses elicited by stimulating DA- or 5-HT-containing neurons or by studying binding properties of DA and 5-HT receptors. These concentrations are on the
scale of 10
6 M for 5-HT, and 10
5 M for DA
(e.g., Ascher 1972
; Fox and Lloyd
1998
; Gospe 1983
; Magoski et al.
1995
; Shozushima 1984
). Thus we believe that the concentrations used in the present study are in the physiological range.
Summary data were represented as means ± SE or as medians with interquartile range (1st quartile to 3rd quartile). The statistical methods of analyses were indicated in the RESULTS and P < 0.05 was taken as indicating significant differences.
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RESULTS |
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DA and 5-HT coordinate different feeding-like movements a semi-intact head preparation
As a first step toward investigating the role of biogenic amines in feeding, it was important to determine the behavioral significance of the DA- and 5-HT-induced activity. To address this issue, we developed semi-intact head preparations in which either DA or 5-HT could be applied and behavior could be measured in quantitative and controllable manner.
Results of pilot experiments
The final design of the semi-intact preparation was an outcome of
extensive preliminary study. First, we assessed the behavioral effects
of DA and 5HT by injecting them into the intact animals (n = 5). These results were unclear. For example, DA
and DOPA induced changes in posture, local skin contractions, and foot disattachment as well as brief movements of the buccal mass. Because the main focus of this study was the modulation of consummatory feeding, we then used a more reduced preparation of head with only
buccal ganglia attached to segregate consummatory feeding component
from the rest of behavioral effects of the transmitters. This approach
was based previous findings suggesting the buccal ganglia contain the
circuit sufficient to generate consummatory feeding (e.g., Kirk
1989; Kupfermann 1974b
; Morton et al.
1991
). In this preparation (n = 4), DA elicited
periodic movements of the buccal mass, but they were weak, did not
appear as bites, or swallows or rejections, and produced only weak
inward displacements of thread placed on radula. Successful feeding
involves coordination of many muscle groups besides the buccal mass
(foot, body wall, and extrinsic buccal muscles), redistribution of
hydroskeleton, etc. In the next set of preparations (n = 4), we retained the cerebral, pedal, and pleural ganglia. We also
canulated the anterior aorta and perfused it under small hydrostatic
pressure to inflate tissues and provide some hydroskeletal support.
When DA was perfused first through the cephalic and pedal arteries,
however, we observed a mixture of effects: local contractions and
withdrawals, penis protrusion as well as abortive feeding movements. We
then adopted the preparation described in METHODS. In this
preparation, the pedal, pleural, and cerebral ganglia were preserved,
but bypassed during perfusion of ASW or monoamines. The advantage of
this preparation was that the isolated head was able to assume a
feeding-like "posture" and exhibit recognizable and strong feeding
movements. Bypassing the cephalic and pedal arteries and perfusing DA
or 5-HT through the buccal artery allowed the drugs to have their
predominate effects on the buccal ganglia. After this preparation was
tested (n = 9), quantitative experiments were performed
in the final setup (Fig. 1).
The role of periphery also was addressed in the pilot experiments (n = 3) in which we removed all the CNS and perfused the head and the buccal mass with DA. No feeding movements were observed; this indicated that the DA-induced activity in the head preparation was not of peripheral origin.
DA induced bite-like movements
Figure 2 compares a bite that was elicited by presenting food (seaweed) to a freely behaving animal (Fig. 2A) to feeding-like movements that were elicited by perfusing a semi-intact preparation with DA (Fig. 2B). In the freely behaving animal, the bite began with the jaws opening to accommodate the protraction of the odontophore (Fig. 2A, 1 and 2). During this initial phase of protraction, the two halves of the radula were open. At the peak of the protraction phase (Fig. 2A3), the two halves of the radula were closed, and they remain closed as the odontophore retracted and the jaws closed (Fig. 2A, 4 and 5).
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DA induced a similar sequence of movements in the semi-intact preparation (Fig. 2B). These movements began with the jaws opening as the odontophore protracted (Fig. 2B, 1 and 2). During this initial phase of protraction, the two halves of the radula were open. At the peak of the protraction phase (Fig. 2B3), the two halves of the radula began to close, and they remained closed as the odontophore retracted and the jaws closed (Fig. 2B, 4 and 5). The recording from the isotonic force transducer for the sequence of DA-induced movements (Fig. 2, B, 1-5, insets) indicated that the thread was drawn into the mouth ~4 mm. Figure 2B, bottom, illustrates the complete record of that experiment from the isotonic force transducer. Before perfusion with DA, feeding-like movements occurred infrequently and there was little net displacement of the thread. Soon after the preparation was perfused with DA, the frequency of feed-like movements increased, and ~13 min into the perfusion, these movements began to produce a net inward displacement of the thread. By the end of the experiment, ~100 mm of thread had been drawn into the foregut and examination of the video record indicated that the jaws opened and closed rhythmically during these feeding-like movements. These results suggest the DA induced biting-like movements.
It should be noted that the effects of perfusing preparations with a
transmitter, such as DA, may not reflect the normal role of the
transmitter. For example, perfusion will activate all receptors for the
transmitter that under normal conditions might not be active together.
To partially address this issue, semi-intact head preparations were
perfused with the metabolic precursor of catecholamines, DOPA.
Precursors, such as DOPA, are presumed to be accumulated selectively
and metabolized by neurons that use the transmitter and to act via
increased release of the transmitter from synaptic terminals of those
cells (e.g., Kabotyanski and Sakharov 1988, 1991
;
Kabotyanski et al. 1994
; McCaman et al.
1984
; Mitchell et al. 1992
). As a result,
precursors affect neural circuitry via endogenous mechanisms, reach
their targets in physiologically relevant order and timing, and produce
gradual and long-lasting effects.
Perfusing with DOPA increased the frequency of feeding-like movements, produced a net inward displacement (i.e., ingestion) of the thread (Fig. 3B), and the DOPA-induced feeding-like movements were similar to those induced by DA (not shown). Both the DA- and DOPA-induced inward displacements of the thread were accompanied by coordinated opening and closing of the jaws as the odontophore rhythmically protracted and retracted (i.e., movements similar to biting in freely behaving animals). The only notable difference between perfusing with DA and DOPA was the greater time required for the feeding-like movements to develop in presence of DOPA. The onsets of sustained feeding rhythm (Fig. 3C1) and net inward displacement of thread (i.e., ingestion; Fig. 3C2) were delayed for ~15 min longer in DOPA than in DA (Fig. 3C). This presumably reflected the time required for dopaminergic cells to accumulate DOPA and metabolize it into DA. Nevertheless a weak agonist also can have delayed effects. If this was the case, the effects would be weaker, however. Yet the effects of DOPA, although delayed, were notably stronger than effects of DA (Fig. 3, A and B). Thus delayed but stronger effects support the hypothesis that DOPA acts as the metabolic precursor of DA and not as a weak agonist.
|
5-HT induced swallow-like movements
Perfusing semi-intact preparations with 5-HT also induced a net
inward displacement of the thread (i.e., ingestion; Fig.
2C). However, 5-HT-induced ingestion was different from DA-
or DOPA-induced movements. As illustrated by the video record in Fig.
2C, 1-4, the jaws remained closed while the thread was
drawn into the mouth (net inward displacement for this sequence was
~10 mm). Figure 2C, bottom, illustrates the complete
record from the isotonic force transducer. Before perfusing with 5-HT,
there was no net change in the position of the thread. Within ~15 min
of perfusing with 5-HT, large inward displacements of the thread were
recorded (i.e., ingestion-like movements). By the end of the
experiment, ~80 mm of thread had been drawn into the foregut. On
average, one 5-HT-induced movement displaced more thread than one
DA-induced bite-like movement (2.730 ± 0.546 vs. 0.614 ± 0.076 mm; 2-sample t-test,
t5 = -3.26, 2-tailed
P < 0.025). There were fewer movements in 5-HT than in
DA (Fig. 3), however, so the resulting average total net displacement
was smaller in serotonin (Fig. 3). Examination of the video record
indicated that the jaws remained closed during these feeding-like
movements. In addition, we could clearly see through transparent
experimental chamber that each inward displacement of the thread was
associated with strong movement of the buccal mass inside the
"head." On the basis of definitions of swallowing during behavioral
observations (Kupfermann 1974b), these results suggest
that 5-HT induced swallow-like movements.
Figure 3 summarizes data from semi-intact preparations that were
perfused with DA, DOPA (Fig. 3, A-C) or 5-HT (Fig.
3D). DA and DOPA had similar effects. First, DA and DOPA
increased the frequency of feeding-like movements from 0.0056 ± 0.0029 to 0.0343 ± 0.0071 Hz and from 0.0086 ± 0.0021 to
0.0452 ± 0.0010 Hz, respectively. Paired t-tests
indicated that these increases were significant (DA:
t2 = 4.54, 1-tailed
P < 0.025; DOPA: t2 =
15.32, 1-tailed P < 0.0025). [As mentioned in the
preceding text, before these experiments, we conducted a series
(n = 17) of pilot experiments using a simplified
design. Thus we had specific hypotheses for the direction of the
effects, which warranted 1-tailed tests used in this section]. Second,
the feeding-like movements that were induced by DA or DOPA produced a
net inward displacement of the thread. Before perfusion with DA, the
average displacement of the thread was 0.3 ± 2.0 mm/h. During
perfusion with DA, the average displacement was 78.8 ± 25.7 mm/h.
Similarly, before perfusion with DOPA, the average displacement of the
thread was
3.2 ± 2.2 mm/h, whereas during perfusion with DOPA,
the average displacement was 117.5 ± 31.6 mm/h. Paired
t-tests indicated that both of these changes were
significant (DA: t2 =
3.11,
one-tailed P < 0.05; DOPA:
t2 =
3.59, 1-tailed
P < 0.04). Although effects of DA and DOPA were
similar in amplitude, the time courses of their development were
different. DA elicited sustained feeding-like movements with a latency
of 59 ± 33 s after the start of perfusion, whereas the effects of DOPA had a latency of 890 ± 18 s (Fig.
3C1). The difference is statistically significant (2-sample
t-test, t4 =
22.09,
1-tailed P < 0.00002). Moreover the number of
feeding-like movements elicited during first 10 min after drug
application was 25 ± 2.6 for DA and 6.3 ± 1.3 for DOPA;
this was significantly different (t4 = 6.30, 1-tailed P < 0.002). Net inward thread
displacement started after 532 ± 130 s of DA perfusion
versus 1,410 ± 206 s after DOPA perfusion (Fig. 3C2;
t4 =
3.60, 1-tailed P < 0.02).
Because the latter effect of DOPA began ~20 min after start of
perfusion, the observation times were ~1 h for DA and 1.33 h for
DOPA. At 1 h, though, the average total net displacements were
similar for the both compounds (90 ± 6.3 mm for DOPA and.
86.5 ± 43.3 mm for DA). This results suggests that DOPA-induced
movements, although delayed, appeared to be more vigorous than
DA-induced movements.
Perfusing semi-intact preparations with 5-HT did not significantly
increase the frequency of feeding-like movements (Fig. 3D1).
In control saline, the average frequency of feeding-like movements was
0.00375 ± 0.00083 Hz, and in the presence of 5-HT, the average
frequency was 0.00667 ± 0.00170 Hz. A paired t-test indicated that this difference was not significant
(t3 = 1.21). In contrast,
perfusing semi-intact preparations with 5-HT significantly increased
the inward displacement of the thread (Fig. 3D2). In control
saline, the average displacement of the thread was
3.7 ± 1.5 mm/h, and in the presence of 5-HT, the average displacement was
60.1 ± 11.2 mm/h. A paired t-test indicated that this
difference was significant (t3 =
5.58, 1-tailed P < 0.006).
The results from the semi-intact preparations indicated that both DA and 5-HT induced and organized ingestion-like movements. However, the results indicated that the two transmitters organized different aspects of ingestion. Bites appeared to be organized by DA, whereas 5-HT appeared to organize swallows. The actions of these two transmitters can result from modulation of either elements in the CNS (e.g., cellular and/or synaptic properties within the CPG) and/or elements in the periphery (e.g., the properties of muscle fibers, release at neuromuscular junctions, and/or sensory feedback). To investigate the neural mechanisms that underlie the actions of these two transmitters, we began by characterizing the motor programs that were expressed in preparations of isolated buccal ganglia.
Characterization of spontaneously occurring BMPs in vitro
Besides the buccal ganglia, other central ganglia play a role in
integrating a correct feeding response in the head preparation, and
their removal may change the parameters of feeding. However, previous
studies showed that rhythmic movements of the odontophore and radula
are mainly supported by the buccal ganglia (e.g., Kupfermann 1974b), and the monoamines in our experiments with the
semi-intact preparation were applied primarily to the buccal ganglia
via the buccal artery. Hence the analysis that follows focuses on the effects of DA and 5-HT on the CPG for rhythmic movements of the odontophore and radula in the isolated buccal ganglia.
During feeding, the rhythmic movements of the radula and odontophore
are produced by coordinated contractions of muscles in the buccal mass,
which in turn, are innervated by motor neurons located in the buccal
ganglia. These motor neurons project to the buccal mass via four
peripheral nerves; the R n. and n.1, n.2, and n.3, respectively (e.g.,
Nargeot et al. 1997; Scott et al. 1991
).
Thus extracellular recordings from these four nerves provide a
comprehensive monitor of centrally generated buccal motor output.
Before investigating the effects of DA and 5-HT on BMPs, we examined
the patterns of neural activity that were generated in preparations of
isolated buccal ganglia, which were perfused with control saline.
Figure 4 illustrates an extracellular
recording of spontaneous rhythmic neural activity from a preparation of
isolated buccal ganglia. A BMP was defined as a sequence of bursts of
large-unit activity in all four buccal nerves. Spontaneous BMPs
occurred most often as discrete, individual patterns (e.g., the neural activity indicated by the box in Fig. 4A), but occasionally
BMPs occurred as a "chain" of multiple cycles of bursting activity (e.g., the neural activity indicated by the bar in Fig. 4A).
Although all BMPs had some features in common (e.g., each began and
ended with a burst of large-unit activity in n.1), several types of BMPs could be distinguished, in part, by the phase relationship between
activity in R n. and n.2. For example, in Fig. 4B, the bursts of large-unit activity in R n. terminated before the burst of
large-unit activity in n.2 (see also Nargeot et al.
1997). This temporal relationship was similar to the neural
correlate of rejection described by Morton and Chiel
(1993a
,b
).
|
In addition to the rejection-like BMP (Fig. 4B), the
isolated ganglia spontaneously expressed several other types of BMPs. A
second type of BMP was characterized by a substantial overlap of
large-unit activities in R n. and n.2 (Fig. 4C) (see also
Nargeot et al. 1997). The temporal relationship was
similar to the neural correlate of ingestion described by Morton
and Chiel (1993a
,b
). A third type of BMP was characterized by
two bursts of large-unit activity in R n. (Fig. 4D). The
first burst in R n. occurred before large-unit activity in n.2 and the
second burst overlapped with large-unit activity in n.2. This temporal
relationship was similar to a pattern that was observed in vivo mainly
during transitions to or from other types of responses (Morton
and Chiel 1993a
,b
). A fourth type of BMP was the chain pattern
(Fig. 4E). The chain BMP had some features similar to those
of the individual BMPs. For example, the chain BMP began and ended with
bursts of large-unit activity in n0.1, and the large-unit activity in R
n. preceded that in n.2. Some differences were observed constantly,
however. First, the chain BMP contained a burst of medium-unit activity in n.1 that overlapped with the burst of large-unit activity in n.2.
Whenever this medium-unit activity was present, the rhythmic activity
continued, and whenever this medium-unit activity was absent, the chain
pattern terminated. Second, the bursts of large-unit activity in n.2
and n.3 did not overlap substantially. At present, the behavioral
relevance of the chain BMP is unknown.
Extracellular recordings indicated that individual preparations
could spontaneously express several different types of BMPs. For
example, the rejection- and transitional-like BMPs illustrated in Fig.
4, B1 and D1, were recorded from the same
preparation. Preparations did not express the different types of BMPs
in equal numbers, however. Figure 5
illustrates the distribution of spontaneously expressed BMPs that was
observed in 55 preparations. A median of 43% [interquartile range
(IR): 9.5-69.5%] of the BMPs were rejection-like, 20% (IR:
0-48.5%) were transitional-like, 0% (IR: 0-21%) were
ingestion-like, and 0% (IR: 0-17.5%) were categorized as other
(e.g., chain patterns). For analyses of the distribution of different
types of BMPs, we used Friedman's testa nonparametric test analogous
to the two-factor ANOVA, repeated-measures design (Zar
1996
). The types of BMPs were the fixed-level factor,
and subjects were the random-effect factor with within subjects'
repeated measures. The test does not depend on normal distribution
assumptions and on scale of measurements. Medians were used to
characterize the populations. The data are ranked and then
2 test statistic is calculated. Friedman's test
indicated a statistically significant difference in the median values
among the four categories (
32 = 29.896, P < 0.001). Post hoc Dunnett's all pairwise
multiple comparison test indicated that preparations of isolated buccal ganglia spontaneously expressed significantly more rejection-like BMPs
than any other type (2-tailed P < 0.05). Thus
although the CPG in the isolated buccal ganglia preparation could
generate several different BMPs, its spontaneous activity appeared to
be biased toward generating the rejection-like BMP.
|
Effects of DA and 5-HT on spontaneous BMPs in vitro
MODULATING THE FREQUENCY OF SPONTANEOUSLY OCCURRING
BMPS.
To determine whether the frequency of spontaneously occurring BMPs
changed over time in vitro, 16 preparations of isolated buccal ganglia
were perfused with control saline for 2 h. The average frequency of
BMPs was 0.0019 ± 0.0003 Hz during the first hour and 0.0021 ± 0.0003 Hz during the second hour (Fig.
6A). A paired
t-test indicated that this change was not significant (t15 =
0.84). Thus the frequency of
spontaneously occurring BMPs was relatively constant for several hours
in vitro. The effects of DA (50 µM) on the frequency of spontaneously
occurring BMPs were examined in 19 preparations (Fig. 6B).
Before application of DA, the average frequency of spontaneous BMPs was
0.00276 ± 0.00049 Hz. During perfusion with DA, the average
frequency increased to 0.00701 ± 0.00109 Hz. A paired
t-test indicated that this increase was significant
(t18 =
4.09, 2-tailed
P < 0.001).
|
|
DA BUT NOT 5-HT BIASED THE SPONTANEOUS OUTPUT OF THE CPG TOWARD
INGESTION-LIKE BMPS.
In addition to increasing the frequency of spontaneously occurring
BMPs, DA modulated the type of BMP that was expressed. Two examples of
this second action of DA are illustrated in Fig. 8. During the 30 min that the preparation
illustrated in Fig. 8A was perfused with control saline,
five spontaneous BMPs were recorded and all five of these were
rejection-like BMPs (e.g., Fig. 8A1). During the 30 min that
this preparation was perfused with DA (50 µM), 11 BMPs were observed
and all of these BMPs were ingestion-like (e.g., Fig. 8A2).
Similar results were obtained in preparations in which simultaneous
extracellular and intracellular recordings were used to monitor BMPs
(Fig. 8B). Figure 8B1 illustrates a
rejection-like BMP that was recorded in control saline, and Fig.
8B2 illustrates an ingestion-like BMP that was recorded from the same preparation after application of DA (50 µM). Recent studies have indicated that high levels of spiking activity in cells B4/5 and
B34 were correlated with rejection-like BMPs (Hurwitz et al. 1997; Kabotyanski et al. 1997
, 1998a
). The level
of spiking activity of B4/5 and B34 appeared to be reduced during the
DA-induced ingestion-like BMP (Fig. 8B2), which suggested
that DA may bias the functional configuration of the CPG toward
generating ingestion-like BMPs by decreasing the excitability of these
cells (see following text).
|
|
Modulation of cellular and synaptic properties by 5-HT and DA
To understand the mechanisms and the role of modulation of the CPG for feeding by DA or 5-HT, we have begun to examine sites in the CPG at which the transmitters might exert their actions. As a first step, it was necessary to characterize the cellular events associated with the action of modulators that occur in the CPG and output neurons under the same pharmacological conditions that led to fictive feeding (i.e., while existing synaptic connections were maintained). Figure 10 illustrates some of the loci within the feeding neural circuitry that were modulated by application of 5-HT. At least three biophysical properties of B31/32 were consistently modulated in the presence of 5-HT (n = 8 preparations). First, the excitability of B31/32 was reduced (Fig. 10A). Second, an oscillation in the resting membrane potential of B31/32 was induced (Fig. 10B). Third, the plateau-like potential in B31/32 appeared to be reduced (Fig. 10C). Similar 5-HT-induced changes were observed in B33 (not shown). In the presence of 5-HT, the membrane potential of B33 oscillated and its excitability was decreased (n = 3 preparations). In contrast to its actions on B31/32 and B33, 5-HT did not induce oscillations or any other observable change in the membrane potential of cell B35. It did, however, appear to increase the excitability of B35 (Fig. 10D) and increased the strength of the B35 synaptic connection to B4/5 (n = 4 preparations). Finally, although the presence of 5-HT did not induce a change in the resting membrane potential of cells B4/5, it did decrease the excitability of these cells and decreased the strength of the B4/5 synaptic connection to B31/32 (Fig. 10E; n = 7 preparations).
|
Some of the same sites that were modulated by bath application of 5-HT also were modulated by presence of DA, albeit in different ways. For example, bath application of DA depolarized the membrane potential of cells B31/32 (n = 7 preparations). This depolarization immediately preceded and appeared to induce the sustained rhythmic activity in the CPG. The depolarizations of membrane potentials in the presence of DA also were observed in cells B8 (n = 8 preparations), B34 (n = 5 preparations), and B65 (n = 2 preparations). In contrast, in the presence of DA, the membrane potentials were hyperpolarized in cells B4/5 (n = 14 preparations), B51 (n = 8 preparations), B63 (n = 2 preparations), and B64 (n = 5 preparations).
Figure 11 illustrates some additional
effects of DA. In the presence of DA, the excitability of cell B34
appear to be reduced (Fig. 11A; n = 6 preparations) and the strength of the excitatory synaptic connection
from B34 to B31/32 was decreased. Similarly the excitability of cells
B4/5 (Fig. 11B; n = 14 preparations) (see also
Kabotyanski et al. 1994, 1997
, 1998a
) was reduced. At the same time, B4/5 exhibited a lower rate of activity in presence of
DA, and in ~20% of preparations, it produced only one to two spikes
per cycle. In addition, bath application of DA decreased the strength
of the synaptic connections from B4/5 to B8 (Fig. 11C;
n = 5 preparations). The excitability of B51 appeared
to be reduced in the sustained presence of DA, and this cell was
usually not active during DA-induced rhythm (not shown;
n = 5 preparations). Although the excitability of B64
was somewhat reduced in the presence of DA (Fig. 11, D and
E; n = 6 preparations), the strength of its inhibitory synaptic connection to B31/32 (Fig. 11D;
n = 3 preparations) and B34 (not shown;
n = 3 preparations) appeared to be increased. In
contrast, the excitatory connection from B64 to B4/5 was reduced in the
presence of DA (Fig. 11E; n = 5 preparations). Finally, bath application of DA led to an increase in
the excitability of B8 and appeared to enhance posthyperpolarization
rebound excitation in this cell (Fig. 11F; n = 10 preparations). Although the actions of DOPA have not been studied
extensively, similar results have been observed. In the presence of
DOPA the excitability of cells B4/5, B34, and B64 appeared to be
reduced. The strength of the inhibitory synaptic connection from B64 to
B31/32 appeared to be enhanced, whereas the strength of the excitatory
synaptic connection from B64 to B4/5 appeared to be reduced.
|
These results indicate that bath application of DA and 5-HT to isolated buccal ganglia elicit diverse and cell-specific changes in identifiable neurons while they are still incorporated in the network. More detailed and quantitative experiments will be necessary to investigate these modulatory actions under conditions that minimize polysynaptic influences.
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DISCUSSION |
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The present study examined the actions of DA and 5-HT in the feeding system of Aplysia at the levels of behavior, neural network activity, and cellular properties. At the behavioral level, both DA and 5-HT induced movements that transported a thread into the foregut of a semi-intact preparation. This result indicated that the DA- and 5-HT-induced movements were ingestion-like. There were several important differences between the features of DA- and 5-HT-induced feeding-like movements, however. First, DA induced feeding-like movements at a high frequency, whereas 5-HT did not alter the frequency of feeding-like movements. Second, the amplitude of individual DA-induced displacements was relatively small as compared with the amplitude of individual 5-HT-induced displacements. Third, DA-induced movements involved opening and closing of the jaws as the radula/odontophore protracted and retracted. In contrast, the jaws remained closed during 5-HT-induced movements. These observations suggested that DA induced bite-like movements, whereas 5-HT induced swallow-like movements.
At the level of the isolated buccal ganglia, 5-HT did not significantly
change frequency of spontaneously occurring BMPs nor did it
significantly change the proportion of ingestion-like BMPs. This latter
result seems to differ from the results in semi-intact preparation in
which we observed strong ingestion induced by 5-HT. The reasons for
this difference are unclear. One possibility is that additional central
and/or peripheral elements may be required for 5-HT to induce neural
correlates of swallowing. For example, to organize swallowing, 5-HT may
require feedback from peripheral structures (e.g., Cropper et
al. 1996; Evans and Cropper 1997
; Jahan-Parwar et al. 1983
; Scott et al.
1995
), and/or 5-HT-mediated modulation of the "physical
plant" may be a key factor in organizing the appropriate movements
(e.g., Chiel and Beer 1993
; Kupfermann et al.
1997
; Weiss et al. 1992
). The absence of the
cerebral ganglion in the in vitro experiments did not seem to be a key
factor in that 5-HT produced similar effects in preparations consisting of isolated cerebral-buccal ganglia (unpublished observations).
The effects of DA at the network level were consistent with its actions
at the behavioral level. Perfusing preparations of isolated buccal
ganglia with DA increased the frequency of BMPs. Moreover the activity
of the CPG was biased toward ingestion-like BMPs by DA. We do not know
whether bath-applied DA reflects the normal role of DA in the buccal
ganglia. Bath application will activate all receptors for DA, which
might not be activated together under normal conditions. To partially
address this issue, we also examined the effects of the DOPA, the
metabolic precursor of catecholamines. The presumed DOPA-induced
elevation of the levels of endogenous DA and exogenous DA induced
similar effects on behavior, neural network activity, and cellular
properties (see also Kabotyanski et al. 1994). These
results suggested that bath application is a reasonable substitute for
the endogenous actions of DA and that DA has at least two roles in the
feeding system. First, DA has an overall activating effect on patterned
motor output of the buccal ganglia, and second, DA modifies the
functional configuration of the CPG such that it generates more
ingestion-like BMPs and fewer rejection-like BMPs.
At the cellular level, bath application of DA and 5-HT had diverse
effects on cells and synaptic connections within the CPG. To evaluate
the functional implications of these changes, however, it is necessary
to understand how the CPG functions and which elements of CPG mediate
different aspects of its function. Recent studies have identified
several cells that mediate specific functions of the CPG, such as cells
that initiate rhythmic activity (e.g., B31/32 and B63) and cells that
shape the patterns (e.g., B4/5, B34, B51, and B65) (Hurwitz and
Susswein 1996; Hurwitz et al. 1994
, 1997
;
Kabotyanski et al. 1997
, 1998a
; Nargeot et al.
1999
, 2000; Susswein and Byrne 1988
). Moreover
recent computational models have begun to reconstruct the circuitry of
the CPG and to explore its function (Baxter et al. 1997
, 1999
,
2000
; Thorne et al. 1997
; Ziv et al.
1994
). These studies provide a conceptual framework within
which the functional consequences of DA- and 5-HT-induced changes in
cellular and synaptic properties can be considered.
Cells that are important for initiating rhythmic activity are targets
for modulation by both DA and 5-HT. Previous studies have indicated
that a key step for initiating a BMP is to sufficiently depolarize
B31/32 so as to elicit a plateau-like potential (Baxter et al.
1997; Hurwitz et al. 1994
, 1997
;
Susswein and Byrne 1988
; Thorne et al.
1997
). 5-HT decreased the excitability and the plateau-like potential of B31/32, which might explain, in part, why the frequency of
BMPs did not increase in 5-HT. Conversely, DA depolarized B31/32, which
might explain, in part, why BMPs were more likely to occur in DA.
Additional factors that may contribute to DA-induced rhythmicity are
the decreased excitability of B64 and the enhancement of its inhibitory
input to B31/32. Simulations studies have indicated that
posthyperpolarization excitation of B31/32 can result from briefer,
more intense B64-mediated inhibition and that this
posthyperpolarization excitation can contribute to genesis of rhythmic
activity (Baxter et al. 1997
, 2000
).
Cells that are important for shaping the patterns of activity are also
targets for modulation by DA and/or 5-HT. Previous studies have
indicated that the levels of activity in cells B4/5 and B34 play
important roles in organizing rejection- versus ingestion-like BMPs
(Baxter et al. 1997; Hurwitz et al. 1997
;
Kabotyanski et al. 1997
, 1998a
). High levels of activity
in B4/5 and B34 appear to contribute to rejection-like BMPs, whereas
low levels of activity favor ingestion-like BMPs. In the present study,
bath application of DA led to a reduction in the excitability and
synaptic strengths of both B4/5 and B34, and B34 was usually not
spiking in the presence of DA, which might explain, in part, why more
ingestion-like patterns and fewer rejection-like BMPs were generated in
DA. The excitability of B4/5 also was reduced by bath application 5-HT,
which might contribute to the ingestion-like feeding movements that
5-HT induced in the semi-intact preparation. In addition, recent
studies have indicated that B51 is another cell that helps to shape
BMPs (Nargeot et al. 1999a
,b
). Activity in B51 was
correlated with ingestion-like patterns in isolated buccal ganglia.
Moreover direct depolarization of B51 during rhythmic activity
increased the number of ingestion-like BMPs, whereas hyperpolarizing
B51 decreased the number of ingestion-like BMPs. In the present study,
bath application of DA led to a hyperpolarization of the membrane
potential of B51 and appeared to reduce its excitability. In addition,
intracellular recordings indicated that B51 generally was not active
during DA-induced rhythmic activity. These results suggest that the
ingestion-like BMPs in which B51 is active may represent neural
correlates of swallowing rather than biting.
Other actions of DA and 5-HT have yet to be explained, in part, because the functions of the cells that they modulate are not well understood. For example, cells B33 and B35 were modulated in the presence of 5-HT, but the roles of these cells in the CPG are unknown. Similarly the presence of DA appeared to enhance posthyperpolarization rebound in B8, but what role this cellular property may play in pattern generation is unknown. Although incomplete, the present analysis is providing links among DA- and 5-HT-induced changes in cellular and synaptic properties to changes in neural network activity and ultimately to changes in behavior.
DA is a possible candidate transmitter for the control of biting
The data of the present study and others suggest that DA plays
important roles in initiating and organizing ingestive behaviors such
as biting. Teyke et al. (1993) characterized a pair of
putative dopaminergic neurons in the buccal ganglia (B20) that were
active during BMPs and that could drive BMPs if depolarized. Moreover, the B20-induced BMPs had several features in common with the BMPs that
are driven by the putative bite-command neuron CBI-2. In addition,
Kabotyanski et al. (1998a)
characterized a second pair of putative dopaminergic neurons (B65) in the buccal ganglia. Depolarization of B65 initiated rhythmic activity and during repetitive activation of B65, the BMPs began to express ingestion-like features. The results of the present study indicated that DA induced
ingestion-like BMPs in the isolated buccal ganglia and bite-like
movements in the semi-intact preparation. In addition, although bath
application of DA led to a variety of different effects on various
cells, some of the actions of DA appear to operate in synergy
consistent with reconfiguration of the CPG and biasing its activity
toward ingestion. For example, depolarization of B31/32 and B65,
associated with an enhanced inhibition from B64 to B31/32, could help
to increase the frequency of BMPs. In addition, depolarization of B65
in presence of DA indicates that these neurons may be an element of a
positive-feedback mechanism by which DA maintains sustained rhythmic
activity. On the other hand, bath application of DA reduced the
excitability and synaptic strength of cell B34. This cell is believed
to play an important role in organizing rejection-like BMPs
(Hurwitz et al. 1997
), and its inhibition should promote the expression of ingestion-like patterns At the same time, decrease of
excitation from B64 to B4/5 and decrease of excitability of B4/5 could
account for reduced firing in B4/5 during retraction. The reduced
firing, together with decreased inhibition from B4/5 to B8A/B, could
led to disinhibition and firing of B8 during retraction (see also
Kabotyanski et al. 1997
, 1998a
). As a result of
these reconfigurations, the balance of B8 activity shifts toward firing mostly during retraction, which is required for the production of
ingestion-like BMPs.
Although the results described in the preceding text indicate that DA
is sufficient to induce ingestion-like activity, they do not indicate
whether DA is necessary for pattern generation. Recent studies of the
effects of the DA antagonist ergonovine, however, address this issue.
Teyke et al. (1993) reported that ergonovine blocked
B20-induced BMPs at concentrations of 10
7 and
10
8 M. Similarly, we found that ergonovine
(EC50 ~8 × 10
8 M)
blocked rhythmic activity that was elicited via tonic stimulation of an
afferent nerve to the buccal ganglia (n. 2,3) (Baxter et al.
1998
; Nargeot et al. 1999c
). These results
suggest that DA is necessary for pattern generation in the buccal ganglia.
Roles of DA and 5-HT in the feeding systems of gastropod mollusks
The effects of exogenous DA and 5-HT on rhythmic activity have
been investigated in a number of gastropods in addition to Aplysia. In Limax (Wieland and Gelperin
1983), exogenous DA (3 × 10
5 M)
induced rhythmic feeding motor programs in reduced preparations. Moreover the frequency and phase relations of the DA-induced motor programs were similar to lip-stimulated fictive feeding. In contrast, exogenous 5-HT (10
5 M) induced broad excitation
of multiple buccal motor units, but this activity exhibited little or
none of the synchronization found in feeding motor programs. Finally,
application of ergonovine (0.5 × 10
6 M)
blocked both DA- and lip-stimulus-induced expression of fictive feeding. These results suggested that DA was both sufficient and necessary for the genesis of feeding motor programs in
Limax. In Helisoma (Arnett 1996
;
Granzow and Kater 1977
; Quinlan et al. 1997
; Trimble and Barker 1984
),
exogenous DA (10
6 to 10
4 M) induced fictive
feeding in reduced and semi-intact preparations. Moreover, DA
antagonists (i.e., sulpiride or haloperidol) blocked fictive feeding.
Bath-applied 5-HT (10
6 to 10
4 M) also was
reported to induce feeding motor programs, and these motor patterns are
believed to mediate repetitive swallowing. In Helix
(Galanina et al. 1986
), bath-applied DA also has been reported to induce fictive feeding, whereas 5-HT modulated but did not
trigger feeding. In addition, injections of the neurotoxin 5,6-dihydroxytryptamine (5,6-DHT), which pharmacologically ablates serotonergic cells, impaired appetitive but not consummatory phases of
feeding. In Lymnaea (Kemenes 1997
;
Kemenes et al. 1990
; Kyriakides and McCrohan
1989
; Tuersley and McCrohan 1988
), bath applied
DA (10
4 M) induced fictive feeding, whereas 5-HT
(10
4 M) led to abrupt cessation of the rhythm (see also
McCrohan and Audesirk 1987
). The actions of 5-HT
appeared to vary depending on the concentration that was used, however.
Concentrations of 5-HT in the nanomolar range did not induced fictive
feeding but did facilitate the production of feeding motor programs
(Yeoman et al. 1994
), which supports the hypothesis the
5-HT has a modulatory rather than command-like role.
Finally, injections of the neurotoxins 5,6-DHT and 6-hydroxydopamine,
which ablate serotonergic and dopaminergic systems, respectively,
indicated that DA was necessary for a basic feeding response to food to
occur, whereas 5-HT has a predominantly modulatory role in feeding behavior.
These data indicate that DA plays a major role in initiating and organizing consummatory feeding responses in gastropods. Perfusing reduced preparations with DA is sufficient to initiate fictive feeding, and blocking the actions of DA or depleting the nervous system of DA impairs rhythmic activity and feeding. Generally, 5-HT is believed to play a modulatory role in feeding. Perfusing reduced preparations with 5-HT did not reliably induce feeding motor programs. However, the presence of 5-HT (or activity in serotonergic cells) could facilitate rhythmic neural activity and feeding. In addition, results from semi-intact preparations indicated that 5-HT may be important for initiating and organizing swallowing.
In conclusion, the results from the present study illustrated that the
isolated buccal ganglia of Aplysia contain a CPG that can manifest several neural correlates of consummatory feeding behavior. This circuitry is multifunctional and can switch between generating rejection- and ingestion-like BMPs. Initiating rhythmic activity in the CPG and switching among different functional
reconfigurations may be mediated, in part, by the transmitter DA. Bath
application of DA led to modulation of several loci within the CPG and
thereby biased its output toward ingestion-like BMPs. Although the BMPs in isolated buccal ganglia may not account for all aspects of consummatory feeding (e.g., 5-HT-induced swallowing), this reduced preparation does appear to retain many key elements of the feeding circuitry and thus can provide a useful model system for cellular and
biophysical analyses of the functional reconfiguration of pattern
generating circuitry and their modulation by learning (e.g.,
Colwill et al. 1997; Lechner et al. 1997
;
Nargeot et al. 1997
).
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ACKNOWLEDGMENTS |
---|
We thank Dr. R. Nargeot and H. Lechner for comments on an earlier draft of this manuscript; Drs. J. Baxter, W. Frost, and R. Nudo for the loan of equipment; and A. Adams, M. Aguirre, W. Amaya, and Y. Noor for providing technical assistance.
This work was supported by National Institute of Mental Health grant R01 MH-58321.
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FOOTNOTES |
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Address for reprint requests: J. H. Byrne, Dept. of Neurobiology and Anatomy and W.M. Keck Center for the Neurobiology of Learning and Memory, The University of Texas-Houston Medical School, P.O. Box 20708, Houston, TX 77225.
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 20 August 1998; accepted in final form 1 October 1999.
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