Department of Neurobiology and Anatomy and W.M. Keck Center for Neurobiology of Learning and Memory, The University of Texas-Houston Medical School, Houston, Texas 77030
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ABSTRACT |
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Nargeot, R., D. A. Baxter, G. W. Patterson, and J. H. Byrne. Dopaminergic synapses mediate neuronal changes in an analogue of operant conditioning. Feeding behavior in Aplysia can be modified by operant conditioning in which contingent reinforcement is conveyed by the esophageal nerve (E n.). A neuronal analogue of this conditioning in the isolated buccal ganglia was developed by using stimulation of E n. as an analogue of contingent reinforcement. Previous studies indicated that E n. may release dopamine. We used a dopamine antagonist (methylergonovine) to investigate whether dopamine mediated the enhancement of motor patterns in the analogue of operant conditioning. Methylergonovine blocked synaptic connections from the reinforcement pathway and the contingent-dependent enhancement of the reinforced pattern. These results suggest that dopamine mediates at least part of the neuronal modifications induced by contingent reinforcement.
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INTRODUCTION |
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Dopamine appears to play a critical role in
several examples of learning, including operant conditioning. For
example, injection of dopamine antagonists or destruction of dopamine
neurons produces deficits in operant conditioning. Moreover,
dopaminergic neurons are activated by reinforcing stimuli, and
electrical or pharmacological stimulation of dopaminergic systems in
the brain can mediate reinforcement (for recent reviews see
Ettenberg 1989; Le Moal and Simon 1991
; Schultz 1997
, 1998
). Despite the involvement of dopamine
in reinforcement, little is known about the cellular mechanisms
underlying its effects.
In recent studies we have begun to investigate the cellular changes
induced by contingent reinforcement in an analogue of operant
conditioning of feeding in the isolated buccal ganglia of
Aplysia (Nargeot et al. 1997, 1999a
,b
).
Dopamine is the primary transmitter believed to be released by the
reinforcement pathway (Kabotyanski et al. 1998a
). Thus
in this study we tested whether methylergonovine, which primarily
affects dopaminergic transmission (Ascher 1972
;
Buckett et al. 1990
; Drummond et al.
1980
; Swann et al. 1978
; Teyke et al.
1993
; Wright and Walker 1984
), could prevent the
effects of contingent stimulation of the reinforcement pathway. A
preliminary report of these results appeared in abstract form
(Baxter et al. 1998
).
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METHODS |
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The experimental procedures of this study were similar to those
described previously (Nargeot et al. 1997). Buccal
ganglia were isolated and pinned out in a Sylgard-coated petri dish
containing either control saline [i.e., artificial seawater (ASW)]
alone or ASW containing methylergonovine. Methylergonovine was the only antagonist examined in this study. The solutions were maintained at
15°C in a static bath by means of a Peltier cooling device. Normal
ASW was composed of (in mM) 450 NaCl, 10 KCl, 30 MgCl2(6H2O), 20 MgSO4, 10 CaCl2(2H2O), and 10 Trizma, pH adjusted to 7.4. In some experiments, a solution of ASW containing high concentrations of Ca2+ and Mg2+ (165 mM MgCl2 and
30 mM CaCl2) was used to block polysynaptic pathways
(Byrne et al. 1978). Solutions of methylergonovine
(Sigma Chemical, St. Louis, MO) were prepared in normal or modified ASW immediately before the experiments. The experimenter was not aware of
either the type of the solution (ASW or methylergonovine) or the
concentration (0.1 nM to 1 µM) that was used. Preparations were
bathed in the solutions for
30 min before recordings were made.
Conventional extracellular and intracellular nerve stimulation and
recording techniques were used. Rhythmic motor activity was elicited by
monotonic (2 Hz) stimulation of an afferent nerve (n.2,3) (for details
see Nargeot et al. 1997). This rhythmic activity was
composed of different motor patterns (i.e., pattern I, pattern II, and
intermediate patterns; see Fig. 1). The
paradigm for stimulating the reinforcement pathway (esophageal nerve, E
n.2) in the analogue of operant conditioning was described by Nargeot
et al. (1997)
. In the contingent reinforcement group, beginning with
the first occurrence of pattern I, stimulation of E n.2 was contingent
on occurrences of this pattern. Training continued for 10 min, and a
minimum of five stimulations of E n.2 was required. In the yoke-control group, the stimulation of E n.2 was delivered with the same timing and
parameters as in a paired contingent-reinforcement preparation. The
monotonic stimulation of n.2,3 was stopped at the end of the training
period and restarted for 20 min, beginning 60 min after training. Data
were collected during the last 10 min of this stimulation period.
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RESULTS |
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High concentrations of methylergonovine blocked rhythmic motor patterns
In the isolated buccal ganglia, rhythmic motor patterns (i.e.,
pattern I, pattern II, and intermediate patterns), which were similar
to those observed during consummatory feeding behaviors (e.g.,
ingestion and egestion), were induced by monotonic stimulation of n.2,3
(Fig. 1A). Pattern I was similar to the motor pattern observed during ingestion in freely behaving animals, and pattern II
was similar to motor pattern observed during egestion (Morton and Chiel 1993). We first examined the effect of
methylergonovine on the rhythmic motor program induced by stimulation
of n.2,3.
The frequency of occurrences of motor patterns decreased with increasing concentrations of methylergonovine (Fig. 1, B-D). A dose-inhibition relationship of the effect of methylergonovine on the rhythmic motor pattern indicated that the apparent concentration of methylergonovine that induced a one-half inhibitory effect (IC50) was 8.4 nM (Fig. 1D).
Methylergonovine blocked monosynaptic effects of the reinforcement pathway
Although 1 nM of methylergonovine had very little effect on the
rhythmic activity induced by stimulation of n.2,3, we tested whether
this concentration affected synaptic connections from the reinforcement
pathway (i.e., E n.2) to three identified cells in the buccal ganglia
(i.e., B4/5, B51, and B52). These cells are believed to be part of the
central pattern generator (CPG)-mediating aspects of feeding (Fig.
2). For example, we found that E n.2 made
an apparent monosynaptic connection with B51, a neuron whose properties
were modified by contingent reinforcement (Nargeot et al.
1999a). This connection persisted in solutions that contained high concentrations of divalent ions and had a fast inhibitory component that was elicited by a single stimulus (0.5 ms) to E n.2
(Fig. 2) and a slow excitatory component that was elicited by
high-frequency stimulation of E n.2 (i.e., 10 Hz, 6 s; not shown).
A concentration of 1 nM of methylergonovine reduced both the fast
inhibitory (Fig. 2A) and the slow excitatory components (not
shown) of the E n.2-mediated synaptic potential (n = 5 preparations). We did not examine a full range of concentrations, but a
high concentration (1 µM) of methylergonovine abolished these E
n.2-mediated synaptic potentials (Fig. 2B; n = 2 preparations).
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Synaptic connections from E n.2 that persisted in solutions containing high concentrations of divalent ion were also observed in cells B4/5 and B52 (Fig. 2C). Methylergonovine (1 µM) abolished the E n.2-mediated synaptic potentials in B4/5 (n = 3 preparations) and B52 (n = 2 preparations). We did not examine the effects of lower concentrations of methylergonovine on these synaptic connections. In all cells examined (i.e., B4/5, B51, and B52) the effects of methylergonovine (1 µM) were only partially reversed by prolonged washing (>1 h) with control saline. B64 is another CPG neuron that received apparent monosynaptic input from E n.2, but the effect of methylergonovine on the E n.2-mediated synaptic potential in this cell was not examined.
A previous study (Wright and Walker 1984) found that in
some molluscan neurons ergonovine could block serotonin (5-HT)- induced hyperpolarizations but not 5-HT-induced depolarizations. To determine whether the E n.2-mediated hyperpolarization of B51 may be mediated via
5-HT, we investigated whether exogenous application of 5-HT mimicked
the actions of the E n.2 and hyperpolarized B51. In three preparations
bath application of 5-HT (5 µM) induced only a slight depolarization
(1 ± 1 mV; values are means ± SE) of the resting membrane
potential of B51. In contrast, bath application of dopamine mimicked
aspects of E n.2 stimulation and hyperpolarized B51 (Kabotyanski et al. 1998b). The results indicated that 5-HT is unlikely to mediate the inhibitory actions of E n.2 on B51 and that low
concentrations of methylergonovine affect the efficiency of the
reinforcement pathway. Moreover, these results suggested that dopamine
may be part of the reinforcing processes.
Methylergonovine blocked contingent-dependent enhancement of motor patterns
To investigate whether methylergonovine modified the effect of
contingent reinforcement in an analogue of operant conditioning, we
used four groups of preparations (Fig.
3). Two groups were bathed in 1 nM
methylergonovine (Fig. 3B). In a contingent-reinforcement group, stimulation of E n.2 was contingent on pattern I during the
training period (for details see Nargeot et al. 1997).
In a yoke-control group, timing of the stimulation of E n.2 was
determined by a paired contingent-reinforcement preparation, and there
was no contingency with the ongoing patterns in the yoke-control
preparation. Two additional groups received the same stimulation
paradigms (contingent reinforcement and yoke control) but were bathed
in control saline (Fig. 3A). The number of occurrences of
pattern I (i.e., the reinforced pattern) during a 10-min test period
beginning ~1 h after training was compared between groups.
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Statistical comparisons (i.e., two-way analysis of variance) indicated a significant difference in the effects of the training paradigms (F = 4.671, df = 40, P < 0.05) that depended on the type of solution used (F = 4.671, df = 40, P < 0.05) (Fig. 3). Post hoc pairwise comparisons indicated that in control saline the number of occurrences of pattern I was significantly enhanced in the contingent-reinforcement group compared with the yoke-control group (q2 = 4.322, P < 0.005; Fig. 3A). In contrast, in the presence of methylergonovine contingent reinforcement did not increase the occurrences of pattern I (Fig. 3B). The contingent-reinforcement paradigm also significantly enhanced the number of occurrences of pattern I in control saline compared with that in methylergonovine (q2 = 4.171, P < 0.01; Fig. 3). Moreover, as expected, in the absence of contingent reinforcement and because of the apparent absence of effect of 1 nM methylergonovine on the ongoing rhythmic activity, no significant change was observed between yoke-control groups in either solutions or in the number of occurrences of the nonreinforced patterns (e.g., pattern II and intermediate patterns) of the four different groups (F = 0.008, df = 40).
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DISCUSSION |
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The results indicated that methylergonovine has at least three effects in the buccal ganglia of Aplysia. First, sufficiently high concentrations of methylergonovine (1 µM) disrupted rhythmic buccal motor patterns induced by monotonic stimulation of n.2,3. Second, a low concentration of methylergonovine (1 nM) reduced E n.2-mediated synaptic potentials, whereas a higher concentration (1 µM) blocked synaptic connections from this reinforcement pathway. Third, a lower concentration of methylergonovine (1 nM) blocked the enhancement of motor patterns induced by contingent stimulation of E n.2 in an analogue of operant conditioning.
In various gastropod mollusks, ergot alkaloids and their derivatives
have been shown to have several different effects, including inhibiting
the binding of dopamine and to a lesser extent 5-HT to receptors
(Drummond et al. 1980); acting as a dopamine agonist and
partial agonist (Gospe and Wilson 1981
; Ku and
Takeuchi 1983
, 1986
; Miyamoto et al. 1979
,
1980
); and acting as dopamine antagonist and mixed antagonist
(Buckett et al. 1990
; Gospe and Wilson
1981
; Juel 1983
; Lukyanetz and Kostyuk
1996
; Pasic et al. 1987
; Wright and
Walker 1984
). Some reports indicate that these agents have no
effect on dopaminergic transmission (Magoski et al.
1995
) or dopamine-stimulated adenylyl cyclase activity
(Deterre et al. 1986
; Yamane and Gelperin
1987
). Although the pharmacological actions of methylergonovine
were not characterized extensively in Aplysia,
methylergonovine is believed to act as a dopamine antagonist at some
dopamine receptors (Ascher 1972
; Gospe and Wilson
1981
; Swann et al. 1978
; Teyke et al.
1993
). This study did not examine the actions of
methylergonovine on exogenous dopamine, but the results did illustrate
that methylergonovine blocked the synaptic inputs from a neural pathway
(i.e., E n.) previously shown to stain positively for dopamine
(Kabotyanski et al. 1998a
). Thus our results suggest
that dopamine plays an important role in both the genesis of feeding
behavior and learning-induced changes in this behavior.
Several lines of evidence support the conclusion that dopamine plays an
important role in the genesis of feeding behavior in
Aplysia. First, several putative dopaminergic cells were
characterized in the neural circuitry that mediates feeding in
Aplysia (Kabotyanski et al. 1998a;
Rosen et al. 1991
; Teyke et al. 1993
).
These cells express rhythmic activity during fictive feeding. Second,
dopaminergic cells were found to participate in the CPG in isolated
buccal ganglia, and direct depolarization of these dopaminergic cells can drive buccal motor patterns that were associated with feeding (Kabotyanski et al. 1998a
; Teyke et al.
1993
). Third, application of exogenous dopamine (or its
metabolic precursor) induces feeding-like movements in semi-intact
preparations (Kabotyanski et al. 1995
) and
feeding-related motor patterns in isolated buccal ganglia (Baxter et al. 1995
). Thus, together with the
observation that methylergonovine blocked rhythmic motor patterns in
the buccal ganglia, these results indicate that dopamine is one of the
key transmitters used by the feeding circuit in Aplysia.
Similar roles for dopamine in feeding were suggested in other
invertebrates and vertebrates (Berry and Cottrell 1973
;
Evans and Eikelboom 1987
; Kemenes 1997
;
Kemenes et al. 1990
; Kyriakides and McCrohan 1989
; Martel and Fantino 1996
; Orosco et
al. 1995
; Sweeney 1963
; Terry et al.
1995
; Weiland and Gelperin 1983
; Wise and
Raptis 1986
; Zhou and Palmiter 1995
).
In addition to playing a role in the genesis of rhythmic activity, this
study suggests a second role for dopamine in feeding. Specifically, it
appears to mediate the reinforcement during operant conditioning.
Several lines of evidence support this conclusion. First, the results
of histofluorescent studies suggest that dopamine is the primary
neurotransmitter in the reinforcement pathway (Kabotyanski et
al. 1998a). Second, apparent monosynaptic effects from the reinforcement pathway were affected by a dopamine antagonist. Third,
this antagonist suppressed the enhancement of motor patterns induced by
contingent reinforcement. This latter effect is unlikely to result from
an action of methylergonovine on the genesis of the rhythmic motor
activity because this activity was not significantly different in
control saline and methylergonovine in the yoke-control groups (Fig.
3). Moreover, no modification was observed for the number of
nonreinforced patterns in the different groups. Finally, the
dose-inhibition relationship of methylergonovine on the rhythmic activity indicated that 1 nM of methylergonovine, which was used to
block the contingent-dependent enhancement, had virtually no effect on
the genesis of the rhythmic activity (e.g., Fig. 1). The different
sensitivity to methylergonovine of the dopamine-dependent processes
that mediate the genesis of rhythmic activity and reinforcement suggests that different types of dopamine receptors are involved. An
important goal for future research will be to identify and characterize
the presumptive dopamine-containing neurons in the reinforcement
pathway and examine the mechanisms by which dopamine exerts its effects
on postsynaptic target cells such as B51.
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ACKNOWLEDGMENTS |
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We thank F. D. Lorenzetti for helping us examine the effects of serotonin on the resting membrane potential of cell B51.
This research was supported by the Ernst Knobil Fellowship, Grant 011618-048 from the Texas Higher Education Coordinating Board, and National Institute of Mental Health Grant MH-58321 and Award K05 MH-00649.
Present address of R. Nargeot: Université Bordeaux I, Laboratoire de Neurobiologie des Réseaux, Bât. Biologie Animal-Bz, Avenue de Facultés, 33405 Talence Cedex, France.
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FOOTNOTES |
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Address for reprint requests: J. H. Byrne, Dept. of Neurobiology and Anatomy, W. M. Keck Center for 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 3 August 1998; accepted in final form 10 December 1998.
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REFERENCES |
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