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Institute of Neurobiology and Department of Anatomy, University of Puerto Rico, San Juan, Puerto Rico
Submitted 15 March 2006; accepted in final form 26 May 2006
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ABSTRACT |
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INTRODUCTION |
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; Winfree 1980). In nervous systems, these capabilities are thought to reflect properties of populations of pulse-coupled nonlinear oscillators (Buzsáki and Draguhn 2004; Hopfield 1994; Singer 1999). To date, opportunities to examine transitions from asynchrony to synchrony in identified neurons with known functions have been limited.
Central pattern generator (CPG) circuits frequently produce alternating movements of bilateral limbs, appendages, or axial muscle systems (Cohen et al. 1988; Grillner et al. 2005; Stein et al. 1997). However, these motor networks can also generate unilateral movements and are often capable of producing synchronous activation of paired effectors (Grillner 1985; Kelso et al. 1979; Stein 2005). Considerable interest is presently focused on understanding how CPG circuits can specify such distinct classes of actions from an individual effector system (Getting 1989; Marder and Calabrese 1996; Stein et al. 1997).
One well-documented means to achieve motor system reconfiguration is via the implementation of neuromodulation (Harris-Warrick and Marder 1991; Kupfermann 1979). Acting as intercellular messengers, neuromodulators can orchestrate coherent modifications of a motor circuit by producing broad and coordinated actions on the synaptic connectivities and intrinsic membrane properties of its constituent neurons (Kiehn and Katz 1999; Marder and Weimann 1992; Parker and Grillner 1998). The actions of neuromodulators are best understood in instances where they initiate activity in CPGs or modify their frequency, phasing, or intensity. The role of modulators as specific regulators of rhythmicity and synchrony of CPG elements has received less inquiry.
Neuromodulation is prevalent in the motor circuits that control the consummatory feeding behaviors of Aplysia (Kupfermann et al. 1979, 1997). The actions of neuromodulators have been exceptionally well characterized in certain neuromuscular components of the feeding system where they typically function as cotransmitters that are released from motor neurons (Brezina et al. 2003a,b, 2005; Weiss et al. 1993; Whim et al. 1993). Although numerous observations indicate that neuromodulators also operate within the central circuits that govern feeding (e.g., Kabotyanski et al. 2000; Kirk et al. 1988; Sossin et al. 1987), our present understanding of their functional capabilities within the buccal CPG is incomplete.
This investigation examined the generation and regulation of firing patterns in B67, an identified bursting motor neuron that innervates the salivary duct and additional buccal muscles in Aplysia californica (Park et al. 1999, 2000). The bursting of B67 was found to reflect properties of an endogenous TTX-resistant sustained driver potential. Under control conditions, B67 bursting was not rhythmic and the activation of the bilateral B67 neurons was asynchronous. The neuromodulator dopamine (DA), however, conferred both rhythmicity and bilateral synchrony to the bursting of B67. A source of direct dopaminergic synaptic innervation of B67 was identified, indicating that the observed DA effects may reflect actions that occur during the physiological operation of this system.
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METHODS |
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Experiments were conducted on specimens of Aplysia californica (150–250 g) that were purchased from the Aplysia Resource Facility and Experimental Hatchery (University of Miami, Coral Gables FL) or from Marinus (Long Beach CA). Animals were maintained in refrigerated aquaria (14–16°C) and fed dried seaweed twice per week.
Electrophysiology
Neurons were identified in preparations consisting of the paired buccal and cerebral ganglia. Intracellular microelectrodes filled with 2 M KCl (10–20 M
) were used for recording. B67 is a large superficial motor neuron located on the caudal surface of each buccal hemiganglion (Park et al. 1999, 2000). Sufficient criteria have been fulfilled to conclude that a neuron tentatively labeled B58 in a preliminary report (Serrano and Miller 2004) corresponds to B67. This cell shares several characteristics with a motor neuron previously designated the pharynx burster (PB) in A. kurodai (Nagahama and Takata 1987). Both neurons innervate multiple muscles that are thought to contribute to swallowing movements. An independent microelectrode (5–10 M) was used for injecting current into B67 (Fig. 1A). B65 is an intrinsic buccal interneuron located within the confluence of the salivary nerve and buccal nerve 1 (Fig. 1A) (Kabotyanski et al. 1998). It contains markers for catecholamines (Díaz-Ríos et al. 2002; Kabotyanski et al. 1998) and GABA (Díaz-Ríos et al. 2002; Jing et al. 2003). Intracellular stimulation of B65 was achieved with the recording electrode across the balanced bridge circuit of the amplifier (NeuroProbe 1600, AM Systems).
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) was used to attenuate polysynaptic activity. Pharmacology
Solutions of drugs were prepared from powder immediately before application. In one series of experiments, the concentration dependence of dopaminergic actions was determined with the antioxidant ascorbic acid (1 mM) present in the solution. Ascorbic acid alone had no detectable effects on B67. Dopamine, sulpiride, and tetrodotoxin (TTX) were obtained from Sigma Chemical (St. Louis, MO). Preparations were superfused with the ASW solution at a rate of 0.5 ml/min using a gravity-fed multi-channel system (ALA Scientific Instruments, Model VM4). Initial exposure to dopamine often initiated a period of coordinated fictive buccal motor programs (see Teyke et al. 1993). When observed, this effect was transient, subsiding within 2 min (see Kabotyanski et al. 2000). In contrast, effects on B67 were robust, and persisted throughout long periods of exposure to DA. As B67 is recruited into coordinated buccal motor programs (Fig. 1B) (see also Park et al. 1999), initial periods of DA exposure were excluded from the present analysis.
Dopamine was also pressure ejected through a puffer pipette (0.5- to 1-s pulses) using a Picospritzer II system (General Valve). In some experiments, the neurotransmitter (1 mM in the pipette) was mixed with a small amount of dextran fluorescein dye to aid visualization of the puff. Application of dextran fluorescein by itself did not produce detectable effects.
Dye injection
After neuron identification, the KCl microelectrode was withdrawn and replaced with one containing Neurobiotin. Injections were modified from the methods described by Delgado et al. (2000). The microelectrode tips were filled with 4% Neurobiotin (Vector Laboratories, Burlingame CA) dissolved in 0.5 M KCl and 50 mM Tris (pH 7.6). The electrode shafts were filled with 2 M KCl, resulting in resistances ranging from 15 to 30 M. Depolarizing current pulses (1–2 nA; 0.5 s; 1 Hz; 10–30 min) were used to eject the Neurobiotin. This procedure did not appear to affect the resting potential or spontaneous electrical activity of the injected neuron. The preparations were usually left at room temperature for 2–3 h to allow material to diffuse from the injection site (cell body) into small and distant processes. They were then repinned if necessary, and fixed in 4% paraformaldehyde (1–4 h). The fixed ganglia were transferred to microcentrifuge tubes, and washed five times (30 min each) with a phosphate buffer containing 1% Triton X-100 and 0.1 mM sodium azide (PTA solution). They were then incubated in Alexa Streptavidin 546 (Molecular Probes, Eugene, OR) diluted (1:800 to 1:3,000) in PTA (24–48 h, room temperature). Tissues were washed five times with PTA and viewed on a Nikon Eclipse TE200 fluorescence microscope prior to immunohistochemistry processing.
Immunohistochemistry
Standard whole mount immunohistochemical protocols were followed (see Miller et al. 1991, 1992 for details of buffer composition, incubation and wash procedures). Ganglia were washed (5 times, room temperature with agitation) in PTA. After preincubation with normal goat serum (0.8%), tissues were immersed (48 h, room temperature) in the primary antibody. A mouse monoclonal antibody (Diasorin, Stillwater MN) generated against rat tyrosine hydroxylase was used at concentrations ranging from 1:50 to 1:200 (see Díaz-Ríos et al. 2002). After repeated PTA washes (5 times, 
30 min each, room temperature), ganglia were incubated in second antibodies conjugated to fluorescent markers (Alexa 488 goat anti-mouse IgG (H+L) conjugate; Molecular Probes: A-11029). The second antibody dilutions ranged from 1:400 to 1:2,000.
Preparations were viewed on the Nikon Eclipse or on a Zeiss Pascal laser scanning confocal microscope (LSCM). Images were captured with the Nikon ACT-1 (Version 2.10) software of the Eclipse or the Zeiss LSM 5 Image Browser (Version 3.1.0.11 [EC] ) program of the Pascal. They were transported as BMP files to Adobe Photoshop for adjusting overall contrast and brightness and then imported to Corel Draw 8 for addition of labels and organization of panels.
Data analysis
All results reported in this study were observed in a minimum of three specimens. Measurements are reported as the means ± SE unless noted. Statistical tests (Student's t-test, 2-tailed) were performed by comparing measurements obtained prior to drug application to those attained at the peak of the response. Multiple group comparisons were performed with the one-way ANOVA followed by Tukey-Kramer pair-wise comparisons. A value of P < 0.05 was established as the criterion for significance. Autocorrelation and cross-correlation functions were generated from occurrences of bursts using the methods applied to spike trains by Perkel et al. (1967a,b). For our analysis, each burst was treated as a point event, the timing of which was assigned at the peak of its initial impulse. To enable quantification of synchronization, an operational index of synchrony, Is, was defined as the fraction of bursts that exhibited partial or temporal overlap of impulse firing. Correlations were displayed graphically using the NeuroExplorer (Version 3.122) software package.
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RESULTS |
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Under control conditions, B67 produced multiple distinct burst patterns. As reported by Park et al. (1999, 2000), B67 was recruited into buccal motor programs (BMPs), where it fired during the protraction phase (Fig. 1B). During the retraction phase of BMPs, B67 received strong inhibition that was usually followed by a rebound burst of impulses.
B67 has also been reported to produce spontaneous bursting (Park et al. 1999). Such spontaneous bursts (2 marked by asterisks in Fig. 1B) were the subject of this investigation. They were highly stereotyped and were not preceded by detectable excitatory or inhibitory synaptic potentials. Each spontaneous burst consisted of 22.7 ± 0.9 action potentials that occurred within a period of 1.15 ± 0.05 s (n = 75 bursts recorded from 12 B67s in 7 preparations). In a given preparation, the burst properties of the two B67s were similar. Their durations (measured as the time from the first to the last impulse) exhibited little variation over a wide range of interburst intervals (IBIs; Fig. 2A). The number of spikes within each burst, however, was dependent on the IBI that preceded it with greater numbers of impulses occurring in bursts that followed briefer IBIs (Fig. 2A). Within the B67 burst, plots of the interspike intervals (ISIs) revealed a U-shaped form, with higher instantaneous spike frequencies (>90/s) occurring near the midpoint of the burst (Fig. 2B). Impulse amplitudes were inversely related to the spike frequency with minimal amplitudes also occurring near the mid-point of the burst (Fig. 2B).
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In contrast to its stereotyped intra-burst firing pattern, the intervals between B67 bursts were highly variable (Fig. 3A). In 12 B67s recorded from seven preparations for a minimum of 1 h (n = 1,116 IBIs), IBIs ranged from 12 to 1,312 s. Depicting the IBIs of a representative pair of B67s as histograms (Fig. 3B) illustrated their broad range with peaks at or near their minimum values (15–40 s). Sometimes additional minor peaks appeared to occur at multiples of the minimal value (Fig. 3B, right).
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20 s in the example shown (Fig. 3C). No additional peaks were observed. Paired recordings of the bilateral B67s typically revealed asynchronous bursting (Fig. 3A). Cross-correllation functions constructed from bilateral pairs disclosed a weak tendency toward synchrony (Fig. 3D). In five B67 pairs, the index of synchrony (Is, see METHODS) ranged from 0.10 to 0.37 (.19 ± 0.11, mean ± SD).
Slow potentials and signaling in B67
Signaling between the two B67s was tested to examine their possible reciprocal influence over bursting. In high-divalent solutions, large hyperpolarizing pulses (40–80 mV) injected into one B67 produced small (1–3 mV) hyperpolarizations in its contralateral counterpart (Fig. 4A). Bursting in one B67 had comparable small (1.3 ± 0.9 mV; n = 8) and long-lasting (2.4 ± 0.4 s) depolarizing effects on the contralateral B67 (Fig. 4B). Deflections corresponding to the impulses were not detected and the peak of this depolarization appeared to occur during the late phase of the contralateral burst (Fig. 4B, dotted vertical lines).
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, 1990). The presence of a driver potential in B67 was tested by blocking impulse activity with tetrodotoxin (TTX). Application of increasing TTX concentrations, beginning at 10–8 M, revealed that fairly high concentrations (5 x 10-6 to 1 x 10–5) were required to affect impulse activity in B67 (cf. Geduldig and Junge 1968; Kado 1973). This concentration blocked nearly all of the B67 spikes in somatic and nerve recordings (Fig. 4C). When B67 spikes were observed, they occurred during the onset of a prolonged TTX-resistant depolarization. In six B67s examined, the duration of this sustained depolarization, measured from the times during onset and decay at which it reached 0.1 of its maximal value, was 2.6 ± 0.3 s. Its peak amplitude, measured from the resting Vm was 25 ± 0.6 mV and its time to peak was 1.28 ± 0.3 s. The parameters of this TTX-resistant persistent depolarization were indicative of the presence of a burst-forming driver potential in B67. Each occurrence of a driver potential was accompanied by a small depolarizing response in the contralateral B67 (Fig. 4D). As was observed with the responses produced by bursts (Fig. 4B), the peak of the contralateral depolarization corresponded to the late phase of the DP (Fig. 4D). Effects of dopamine on B67 burst properties
The patterned bursting of B65 was modified by dopamine, a major regulator of feeding programs in Aplysia (Kabotyanski et al. 1998, 2000; Nargeot et al. 1999; Teyke et al. 1993). The concentration dependence of dopaminergic actions was tested by exposing the ganglion to increasing bath concentrations of DA with an antioxidant (ascorbic acid, 1 mM) included in all solutions (Fig. 5A). Threshold concentrations were in the micromolar range for dopamine-induced increases in the B67 burst duration and impulses per burst. The dose-response function for both parameters was rather steep with maximal effects occurring in the range of 5 x 10–5 to 1 x 10–4 M.
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The actions of dopamine on B67 bursting were examined in more detail with concentrations that produced maximal effects (Fig. 6). Application of DA at 10–4 M increased the burst duration from 0.88 ± 0.09 to 1.44 ± 0.13 s (t = 2.57, P < 0.05, n = 6; Fig. 6B1) and the number of impulses per burst was increased from 26.2 ± 2.0 to 46.2 ± 1.3 (t = 2.02, P < 0.05, n = 6; Fig. 6B2). The mean frequency of impulse firing within the bursts was not affected by dopamine (control: 31.2 ± 3.5 spike/s; DA: 35.7 ± 2.8 spike/s; P > 0.05, n = 6; Fig. 6B3).
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The rate of B67 bursting was also increased in the presence of dopamine (Fig. 8A). With bath application of DA (1 x 10–4 M), the frequency of bursting was rapidly elevated to a higher level that was maintained throughout prolonged exposure (Fig. 8B). Dopamine reduced the mean IBI from 265.8 ± 116.5 to 16.0 ± 1.6 s (t = 2.26, P < 0.05; n = 10 B67s from 6 preparations; Fig. 8C1). The elevated burst frequency appeared to be accompanied by an increase in the regularity of B67 bursting (Fig. 8A). The degree to which DA influenced the uniformity of IBIs was therefore assessed by calculating their coefficient of variation [Cv = (
/µ) x 100], an attribute that normalizes their variability and expresses it as a percentage. The coefficient of variation of IBIs was decreased in the presence of DA from 90.0 ± 11.2 to 8.1 ± 2.9% (t = 2.26, P < 0.05, n = 10; Fig. 8C2).
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Two series of tests were conducted to further clarify the relation between B67 burst rhythmicity and synchrony. As previous experiments had shown that injection of a constant depolarizing current could induce rhythmic bursting (see Fig. 2C), it was possible to test whether such rhythmic activity per se in one B67 could entrain its contralateral companion. Depolarization of either the right B67 (Fig. 10A1) or the left B67 (Fig. 10A2) reduced the IBI coefficient of variation of the injected cell to a level that was significantly lower than its nondepolarized mate (Fig. 10B). In neither instance, however, was the index of synchrony increased over control values (Fig. 10C). The Is observed with depolarization of either B67 remained significantly lower than that induced by dopamine (Fig. 10C).
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The limited number of catecholaminergic neurons in the buccal system (Díaz-Ríos et al. 2002; Kabotyanski et al. 1998; Teyke et al. 1993) enabled a search for possible sources of dopaminergic synaptic innervation of B67. Dye fills of one known dopaminergic interneuron, B65, revealed that it possessed suitable morphological properties for the production such signaling (Fig. 13). In agreement with previous observations (Kabotyanski et al. 1998), B65 was found to project its major process through the central core of the ganglion (Fig. 13A1) to the contralateral hemiganglion. In both hemiganglia, this process passed in close proximity to B67 (Fig. 13, A and B). The soma and principal process of B65 gave rise to numerous secondary and tertiary collaterals that coursed toward the motor neurons of both hemiganglia (Fig. 13, A2 and B2) (see also Kabotyanski et al. 1998). Double-labeling experiments using an antibody to tyrosine-hydroxylase (TH), revealed TH-like immunoreactive fibers immediately adjacent to both B67s (Fig. 13, A1 and B1).
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; Kabotyanski et al. 1998) and that its rapid excitatory signaling to several follower neurons is mediated by dopamine (Díaz-Ríos and Miller 2005; Due et al. 2004). Activation of the receptors that generate these excitatory postsynaptic potentials (EPSPs) can be accomplished with rapid focal application of high concentrations of DA (1 mM) (see Díaz-Ríos and Miller 2005; Due et al. 2004). As bath-applied DA (
1 x 10–4 M) did not affect the membrane potential or input resistance of B67 (Fig. 5B), puffed application from a micropipette was used to probe for such receptors (Fig. 14A). A brief (200 ms) pulse of DA (1 mM in the pipette) to the soma-initial segment of B67 produced a depolarizing response that could exceed threshold (Fig. 14A, top). This depolarizing response was reversibly blocked by sulpiride (1 mM), a known antagonist of dopaminergic synaptic receptors in mollusks (Magoski et al. 1995; Quinlan et al. 1997) (Fig. 14A, middle and bottom). These findings indicate that excitatory dopaminergic synaptic receptors are present on B67.
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Finally tests were conducted to assess the ability of B65 to promote rhythmicity and synchrony of B67 bursting. Bursts of B65 generated by injection of depolarizing current pulses (5 s) produced prolonged periods of bilateral bursting in B67 (Fig. 14E). The bursting during this period, which could last several minutes, was highly rhythmic. Finally, the bursting of the two B67s after firing of a single B65 was highly synchronized. It is proposed (see DISCUSSION) that modulatory dopaminergic signaling originating from B65 can produce the observed extended periods of rhythmic synchronous bursting in B67 (Fig. 14F, see DISCUSSION).
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DISCUSSION |
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; see also Nagahama and Takata 1987). We suggest that its activation by dopamine can originate from B65 during feeding and that it serves to coordinate increases in salivation and buccal movements that occur during consummatory behaviors (Fig. 14F). Synchronous salivation is postulated to reflect the adaptive value that simultaneous bilateral lubrication of the pharynx and esophagus would confer on the ability to ingest or egest large objects. Moreover, simultaneous bilateral contraction of buccal muscles could provide more effective forces for translocating objects that span the esophagus.
In agreement with previous reports (e.g., Kabotyanski et al. 2000), spontaneous buccal motor programs occurred very infrequently (once per 5–10 min) in the in vitro cerebral-buccal preparation used in this study. Although B67 was recruited into such programs (Fig. 1B) (Park et al. 1999; see also Prior and Gelperin 1977), the bursting analyzed in this investigation occurred at much higher frequencies (see Fig. 3). Although we propose that its frequency is influenced by consummatory programs (Fig. 14F), this bursting was not driven on a cycle-to-cycle basis by the buccal CPG that generates well-characterized ingestive and egestive BMPs (Cropper et al. 2004; Elliott and Susswein 2002). This form of bursting is therefore not likely to produce movements that are readily detected with behavioral observation, such as biting and rejection (Kupfermann 1974; Morton and Chiel 1993; Rosen et al. 1989). We propose that, in common with these coordinated motor programs (Kabotyanski et al. 1998, 2000) the activation of B67-driven movements can be achieved by dopamine release from B65 (Fig. 14F).
Endogenous properties of B67
B67 exhibited features that contribute to pattern and rhythm generation in motor systems (Friesen 1994; Hartline et al. 1988; Pearson 1993). Several neurons in the Aplysia feeding network exhibit endogenous burst-forming potentials or bistable states (Perrins and Weiss 1998; Plummer and Kirk 1990; Susswein and Byrne 1988). Prolonged, regenerative, TTX-resistant potentials were originally identified in the motor neurons of the crustacean cardiac ganglion (Tazaki and Cooke 1979, 1990). They were designated "driver potentials" due to their proposed role in the generation of impulse trains. In agreement with the observations of Tazaki and Cooke (1979), the duration of the B67 driver potential in TTX was substantially longer than the slow potential that underlies normal bursting. In the case of the crustacean motor neurons, such differences in duration could be attributed, at least in part, to the relatively refractory state of the slow calcium current that underlies the DP when evoked at frequencies corresponding to the heartbeat (Tazaki and Cooke 1990). Differences in the duration of the B67 burst and its DP are unlikely to reflect refractory properties of contributing currents, as IBIs typically exceed 15 s. A complete understanding of B67 bursting will require a detailed characterization of the biophysical processes that contribute its driver potential.
Although burst-forming potentials have been described in a range of neural systems (e.g., Calabrese and Peterson 1983; Wallen and Grillner 1987; Wong and Prince 1981), their direct participation in intercellular interactions may be less prevalent. A notable instance of such signaling occurs in the crustacean stomatogastric ganglion, where complex network rhythms can be achieved in the absence of impulses (Anderson and Barker 1981; Raper 1979). The onset of the depolarization produced by a B67 burst in its contralateral counterpart began several hundred milliseconds after the initiation of impulse firing and its peak occurred near the end of the burst. Deflections corresponding to impulses could not be distinguished. This depolarization persisted in TTX, again beginning after a delay and peaking during the late phase of the driver potential. These observations suggest the participation of the slow burst-forming potential in B67-to-B67 signaling. However, the capacity for such interactions remains enigmatic, as potential sites of apposition have not been reported for B67 or the pharynx burster of A. kurodai (Nagahama and Takata 1987; Park et al. 1999; this study).
Modulation of B67 bursting by dopamine
Our observations indicate that dopamine can produce coordinated modifications of several attributes of B67. The DA-induced prolongation of the B67 driver potential was consistent with its ability to increase burst duration. Also, the DA-induced enhancement of signaling between the two B67s corresponded well to its ability to increase their synchrony. The overall effects of DA on B67 are likely to reflect the concurrent and collective modulation of several properties that combine to endow B67 with increased burst duration, frequency, rhythmicity, and synchrony. Similar coordinated actions of dopamine on multiple synaptic and biophysical properties have been exceptionally well documented in the crustacean stomatogastric system where DA modifies burst rate and phase relations (Flamm and Harris-Warrick 1986; Harris-Warrick et al. 1995; Johnson and Harris-Warrick 1990).
The burst properties of B67 were modified by concentrations of dopamine ranging from 10-5 to 10-4 M. These concentrations are comparable with those that have been found to modulate fictive motor rhythms in several CPGs (Kemnitz 1997; Miller et al. 1984; Raper 1979), including the buccal CPG of Aplysia (Kabotyanski et al. 2000). They are substantially lower, however, than those (10-3 M) that are required to activate the receptors that mediate rapid EPSPs produced by dopaminergic buccal interneurons (Díaz-Ríos and Miller 2005; Due et al. 2004). These observations indicate that multiple classes of dopaminergic receptors contribute to the operation of this circuit (see also Nargeot et al. 1999; Teyke et al. 1993). The finding that B67 receives direct rapid dopaminergic EPSPs indicates that at least some regions of its membrane, i.e., those that are subsynaptic or perisynaptic to its innervation from B65, can be exposed to high levels of DA (Fig. 14F).
The effects of firing a single B65 on bursting in B67 could last several minutes and were observed after detectable indications of its direct synaptic signaling had subsided. The proposed prolonged actions of DA would therefore be classified as modulatory (see Kaczmarek and Levitan 1987; Kupfermann 1979). Interestingly, although the rapid dopaminergic synaptic signaling of B65 to several follower neurons has been shown to bias motor programs toward an egestive mode (Due et al. 2004), its longer-lasting signals produce a gradual transition toward ingestive patterns (Kabotyanski et al. 1998). The prolonged duration of B65's actions on the rhythmicity and synchrony of B67 suggests that these effects are more closely related to B65's slower ingestive-promoting influence. If dopamine is responsible for this sustained action then the dopaminergic signaling from B65 would include both rapid EPSPs and this long-lasting modulatory component. A similar form of conjoint rapid and sustained modulatory signaling mediated by a single biogenic amine, serotonin, has been demonstrated in the escape swim CPG of the nudibranch Tritonia (Clemens and Katz 2001; Katz and Frost 1995). In the case of B65, however, it will be necessary to distinguish effects of dopamine on B67 from those of another potential modulator, GABA, as GABA-like immunoreactivity has also been localized to B65 (Díaz-Ríos et al. 2002; Jing et al. 2003) and GABA can modulate its rapid synaptic signaling (Díaz-Ríos and Miller 2005).
The effects of dopamine on B67 are consistent with its demonstrated ability to activate feeding motor programs in mollusks (Quinlan et al. 1997; Trimble and Barker 1984; Wieland and Gelperin 1983) including Aplysia (Kabotyanski et al. 2000). The capacity of additional known sources of dopamine in this system, such as the cerebral-buccal interneuron CBI-1 (Rosen et al. 1991), the buccal-cerebral interneuron B20 (Teyke et al. 1993), and esophageal afferents (Kabotyanski et al. 1998) to induce rhythmicity and synchrony in B67 remains to be explored.
Conditional rhythmicity and synchrony
Most models of spontaneous synchronization in biological systems have been described for large populations of oscillating elements, such as chorusing crickets, flashing fireflies, rhythmogenic myocardial cells, and circadian pacemaker neurons (Kuramoto 1984; Peskin 1975; Winfree 1980). In the bag cell neurosecretory system of Aplysia, bilateral aggregates of 400 neuroendocrine cells, intracluster and bilateral synchrony is thought to reflect a combination of electrotonic and autoexcitatory neurohormonal interactions (Brown and Mayeri 1989; Haskins and Blankenship 1979; Kupfermann and Kandel 1970). In the buccal network examined here, synchronization was achieved in a system composed of a small number, possibly as few as two, constituent units. Moreover, a single modulator originating from an identified source was found to be sufficient for producing the transition from asynchrony to synchrony by this system.
The capability of a neuromodulator to induce bursting in otherwise quiescent motor networks (see Miller and Sullivan 1981; Ramirez and Pearson 1989; Raper 1979) has been designated "conditional bursting" (Harris-Warrick and Flamm 1987; Marder and Eisen 1984). The present observations differ fundamentally from this form of modulation, as B67 produces spontaneous bursts in the absence of dopamine. In keeping with this terminology, however, we refer to the actions of DA on B67 as promoting "conditional rhythmicity" and "conditional synchrony."
In many synchronizing biological systems, synchronization is accompanied by, and may be contingent on, transitions to rhythmicity (Buck and Buck 1968; McClintock 1971; see Strogatz 2003). With respect to the synchrony imposed on B67 by dopamine, this also appears to be the case. Numerous additional neuromodulators are present in the buccal system (Kupfermann et al. 1979; Miller et al. 1993a,b; Whim et al. 1993), so it should be feasible to examine whether other signals can promote synchrony in the paired B67s without inducing their rhythmicity. Likewise, it should be possible to determine whether modulators can generate B67 rhythmicity without producing synchrony. The multitude of modulatory systems that are present in the feeding CPG of Aplysia should provide opportunities to explore relations between rhythmicity and synchrony in an exceptionally tractable neuronal network with well-understood behavioral functions.
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GRANTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. W. Miller, Institute of Neurobiology, University of Puerto Rico, 201 Blvd del Valle, San Juan, Puerto Rico 00901 (E-mail: mmiller{at}rcm.upr.edu)
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; right hemiganglion, 
) and the impulses per burst (left hemiganglion, 
s; right hemiganglion, 
) as a function of interburst interval (IBI). Over the entire extent of IBIs (10–100 s), the burst durations of both B67s remained within a narrow range (duration left = 0.97 ± 0.04 s; duration right = 0.84 ± 0.04 s). At lower IBI values (15 –25 s), however, the number of spikes per burst was dependent on IBI duration with more impulses occurring in bursts following shorter IBIs. With IBIs >25 s, the spikes per burst remained within a narrow range (15–17). B: within the B67 burst, impulse amplitude varied as a function of interspike interval. Action potentials were measured from the point at which they departed from the slow underlying potential to their peaks. The smallest values were measured near the midpoint of the burst, when the instantaneous firing frequency was at its highest level. C: responses of B67 during long-duration (60 s) intracellular current pulses. The IBI decreased when the current amplitude was increased and bursting became more rhythmic (see also 
). This spike arose from the rising phase of a sustained depolarization that reached a peak of 
) and number of impulses per burst (
). Each pair of data points is based on 5 observations. All tests were made with 1 mM ascorbic acid present in the medium to retard oxidation. Representative recordings at 1 x 10–7 and 5 x 10–5 M are from the same preparation. Calibration applies to both recordings. B: effects of bath-applied DA on the B67 burst were not accompanied by detectable changes in the resting membrane potential or input resistance. B1: representative recordings (insets) of a B67 bursts prior to application of DA (Con) and at the peak of a response to 1 x 10–4 M dopamine (DA). In both records, a dotted line is drawn at –50 mV. Current pulses (I) injected prior to DA application and at the peak of the response produced equivalent deflections of the membrane potential. B2: time course of dopamine actions on B67. Bath application of DA (1 x 10–4 M) produced an increase of nearly 200% in the number of impulses per burst (
) measured as in B1.
) were variable prior to DA application but were highly regular in DA. C: summary data illustrating effects of DA on B67 burst regularity. Left: dopamine (1 x 10–4 M) produced a significant reduction of the mean IBI (t = 2.26, P < 0.05; n = 10). Right: dopamine (1 x 10–4 M) produced a significant reduction of the IBI coefficient of variation (t = 2.26, P < 0.05; n = 10).
