Target-Specific Regulation of Synaptic Efficacy in the Feeding Central Pattern Generator of Aplysia: Potential Substrates for Behavioral Pla
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Institute of Neurobiology and Department of Anatomy, University of Puerto Rico, 201 Blvd. del Valle, San Juan, Puerto Rico 00901
Abstract
The contributions to this symposium are unified by their focus on the role of synaptic plasticity in sensorimotor learning. Synaptic plasticities are also known to operate within the central pattern generator (CPG) circuits that produce repetitive motor programs, where their relation to adaptive behavior is less well understood. This study examined divergent synaptic plasticity in the signaling of an influential interneuron, B20, located within the CPG that controls consummatory feeding-related behaviors in Aplysia. Previously, B20 was shown to contain markers for catecholamines and GABA (Díaz-Ríos et al., 2002), and its rapid synaptic signaling to two follower motor neurons, B16 and B8, was found to be mediated by dopamine (Díaz-Ríos and Miller, 2005). In this investigation, two incremental forms of increased synaptic efficacy, facilitation and summation, were both greater in the signaling from B20 to B8 than in the signaling from B20 to B16. Manipulation of the membrane potentials of the two postsynaptic motor neurons did not affect facilitation of excitatory postsynaptic potentials (EPSPs) to either follower cell. Striking levels of summation in B8, however, were eliminated at hyperpolarized membrane potentials and could be attributed to distinctive membrane properties of this postsynaptic cell. GABA and the GABAB agonist baclofen increased facilitation and summation of EPSPs from B20 to B8, but not to B16. The enhanced facilitation was not affected when the membrane potential of B8 was pre-set to hyperpolarized levels, but GABAergic effects on summation were eliminated by this manipulation. These observations demonstrate a target-specific amplification of synaptic efficacy that can contribute to channeling the flow of divergent information from an intrinsic interneuron within the buccal CPG. They further suggest that GABA, acting as a cotransmitter in B20, could induce coordinated and target-specific pre- and postsynaptic modulation of these signals. Finally, we speculate that target-specific plasticity and its modulation could be efficient, specific, and flexible substrates for learning-related modifications of CPG function.
Introduction
The contributions to this symposium underscore the central position of activity-dependent synaptic plasticity in our present understanding of sensorimotor learning. One attribute of such plasticities that renders them suitable for producing adaptive behavior is their own capacity to be modified (Krasne, 1978; Byrne and Kandel, 1996; Abraham and Bear, 1996). Recent investigations have broadened our appreciation for the occurrence of diverse forms of synaptic plasticity within the central pattern generator (CPG) circuits that produce repetitive behaviors (Marder, 1998; Parker and Grillner, 1999, Parker and Grillner, 2000; Nadim and Manor, 2000; Sakurai and Katz, 2003; Yuste et al., 2005). The contribution of such activity-dependent modifications of synaptic strength to adaptive CPG-driven motor activity, however, remains incompletely understood.
The consummatory behaviors of Aplysia are generated by a multifunctional or polymorphic CPG that supports several forms of non-associative and associative behavioral plasticity (Susswein and Schwarz, 1983; Kupfermann et al., 1989; Colwill et al., 1997; Lechner et al., 2000a). Cellular analyses of in vitro analogs have begun to reveal potential correlates of experience-dependent plasticity at multiple levels of the feeding network (Nargeot et al., 1997, Nargeot et al., 1999a, Nargeot et al., 1999b; Lechner et al., 2000b; Brembs et al., 2002, Brembs et al., 2004; Mozzachiodi et al., 2003; Proekt et al., 2004). These correlates often correspond to loci where activity-dependent synaptic plasticity is known to occur. For example, command-like interganglionic projection neurons in the cerebral ganglion activate CPG circuits in the buccal ganglion via excitatory postsynaptic potentials (EPSPs) that exhibit various forms of short-term enhancement (Sánchez and Kirk, 2000, Sánchez and Kirk, 2002; Hurwitz et al., 2003). Signaling from the interneurons within the buccal CPG to the motor neurons that execute consummatory feeding behaviors also displays several forms of plasticity (Teyke et al., 1993; Kabotyanski et al., 1998; Jing and Weiss, 2002). Finally, patterned firing by the motor neurons themselves produce facilitating or depressing excitatory junction potentials in the buccal muscles (Cohen et al., 1978; Cropper et al., 1990; Jordan et al., 1993). The possible involvement of these synaptic plasticities with feeding-related behavioral plasticities is largely unexplored.
Regulation of synaptic efficacy by neuromodulatory messengers also plays a pivotal role in learning and memory in several invertebrate model systems (Kandel and Schwartz, 1982; Hawkins et al., 1993; Menzel, 2001; Balaban, 2002). Neuromodulatory control has been intensively studied in CPG circuits, where broadly acting messengers are capable of regulating the biophysical properties of constituent neurons and their synaptic connections in a coherent fashion (Kupfermann, 1979; Harris-Warrick and Marder, 1991; Katz, 1999). When modulators originate from neurons that are not sensu stricto participants in the CPG, they are considered extrinsic (Kupfermann et al., 1979; Morgan et al., 2000). When they derive from neurons that are themselves elements of the CPG (Katz and Frost, 1996), or from motor neurons (Cropper et al., 1987), they are designated intrinsic. In both configurations, modulators are frequently released from neurons in which they occur as cotransmitters (Kupfermann, 1991; Weiss et al., 1992; Nusbaum et al., 2001).
Recently, colocalization of markers for GABA and catecholamines was demonstrated in B20 (Díaz-Ríos et al., 2002), an intrinsic buccal CPG interneuron that can initiate and specify the consummatory feeding motor programs of Aplysia (Teyke et al., 1993; Jing and Weiss, 2001; Proekt et al., 2004). Pharmacological evidence supported the role of dopamine as the mediator of fast synaptic signaling from B20 to B16 and B8, two motor neurons that cause closure of the food-grasping radula (Fig. 1A; see Díaz-Ríos and Miller, 2005). GABA, acting via GABAB-like receptors, exerted differential modulatory actions on synaptic transmission from B20 to B16 and B8 (Díaz-Ríos and Miller, 2005). In that study, the effects of GABA were tested on EPSPs that were evoked by single B20 impulses. As B20 ordinarily fires in a burst mode during feeding motor programs (Teyke et al., 1993; Jing and Weiss, 2001; Proekt et al., 2004), this investigation explored some plastic properties of its divergent synaptic signaling and their modification by GABA. Some of these observations have appeared in abstract form (Díaz-Ríos and Miller, 2002).
Materials and Methods
Subjects
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, Miami, FL) or from Marinus Inc. (Long Beach, CA). Animals were maintained in a refrigerated aquarium (14–16 °C) and fed dried seaweed twice per week. Experimental protocols followed established guidelines of the Institutional Animal Care and Use Committee of the University of Puerto Rico Medical Sciences Campus.
Electrophysiology and pharmacology
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 to record from B20 and its direct synaptic follower motor neurons B16 and B8 (Teyke et al., 1993). Current was injected into the postsynaptic cells by a second intracellular electrode (5–10 M). B20 was stimulated via passage of current through the recording electrode across the bridge circuit of the amplifier (NeuroProbe, A-M Systems). All experiments were conducted in artificial seawater (ASW) containing high concentrations of divalent cations (Liao and Walters, 2002) to attenuate polysynaptic activity.
Solutions of drugs at the concentration to be applied were prepared in high-divalent ASW immediately before the start of each experiment. GABA and (±)-baclofen were obtained from Sigma Chemical Co. (St. Louis, MO). All experiments were performed with application of agonists at a concentration of 1 mM, which produced consistent and reversible effects in previous studies (see Díaz-Ríos and Miller, 2005). Drugs were delivered at a rate of 0.5 ml/min by a gravity-fed perfusion system (ALA Scientific Instruments, model VM4). Responses were recorded 2, 5, and 10 min after switching the perfusion source.
Measurements and statistics
A measurement of summation (Sumn; unit: mV) for a particular EPSP in a train (EPSPn) was obtained by subtracting the postsynaptic membrane potential prior to initiation of the train (Vrest) from the membrane potential immediately prior to the onset of EPSPn (Fig. 1B). An operational measure of facilitation of EPSPn (Fn, a unitless value) was defined as (EPSPn/EPSP1) – 1, so that a value of 0 signified the absence of facilitation (Fig. 1B). EPSP amplitudes were corrected for summation by subtracting Sumn. They were also corrected for changes in driving force (Martin, 1955), using an estimated value of +5 mV as the reversal potential of B20-evoked fast EPSPs (Díaz-Ríos and Miller, 2005).
In view of the presence of long-lasting forms of plasticity and GABAergic actions in this system (Díaz-Ríos and Miller 2002, Díaz-Ríos and Miller 2005), statistical tests (Student’s paired t test; two-tailed) were performed by comparing measurements obtained prior to drug application to measurements obtained at the peak of the response. A value of P < 0.05 was established as the criterion for significance.
Results
Divergent synaptic signaling from B20 to B16 and B8
In a previous study, we found the rapid synaptic signaling from B20 to B16 and B8 to be mediated by dopamine (See Fig. 1A andDíaz-Ríos and Miller, 2005). During that investigation, repetitive firing of B20 revealed two forms of incremental increases in the efficacy of its excitatory signaling—facilitation and summation (Fig. 1B; see also Teyke et al., 1993; Jing and Weiss, 2001). Simultaneous recording from B16 and B8 allowed us to directly compare facilitation and summation during their responses to B20 impulse trains (Fig. 2A). Facilitation values (see definition in Materials and Methods) measured at the 10th EPSP in a train in B8 (1.8 ± 0.4; mean ± SEM) were greater than those observed in B16 (1.2 ± 0.2; t = 4.63; P < 0.05; n = 4; Fig. 2B). Summation values (see definition in Materials and Methods) measured at the tenth EPSP in a train in B8 (6.1 ± 0.5 mV) were also greater than those observed in B16 (4.3 ± 0.3 mV; t = 7.59, P < 0.05; n = 4; Fig. 2C).
The activity-dependent short-term plasticity of EPSPs originating from B20 was further examined in experiments in which the postsynaptic membrane potentials of B16 and B8 were set to hyperpolarized levels. Synaptic facilitation, which is generally thought to reflect presynaptic conditions that produce increases in neurotransmitter release (del Castillo and Katz, 1954; Fisher et al., 1997; Zucker and Regher, 2002), was not affected by this manipulation (Fig. 2A–C). Summation, on the other hand, was differentially affected in the two synaptic followers. Whereas the measures of summation increased in B16 when that cell was pre-set to hyperpolarized levels (Fig. 3A, D), summation was severely attenuated in B8 when it was hyperpolarized (Fig. 3B, D).
The differential effects of membrane potential on synaptic summation in B16 and B8 prompted us to compare the current-voltage relations in these two motor neurons. Voltage responses to current pulses (2 s) injected into B16 rapidly reached a stable value (Fig. 4A). A plot of these voltage displacements as a function of injected current values revealed a predominantly linear relation with limited rectification in the depolarizing quadrant (Fig. 4A2). Responses to current pulses in B8 were more complex (see also Klein et al., 2000; Díaz-Ríos and Miller, 2005). Pulses to levels more hyperpolarized than about –70 mV exhibited a depolarizing sag (Fig. 4B1, arrow). Upon release from these large pulses, the membrane potential of B8 displayed a transient depolarization (Fig. 4B1, arrowhead) prior to reestablishing its resting level. Although not further explored here, such responses have been shown to reflect the presence of hyperpolarization-activated inward currents (Ih) in other invertebrate motor systems (Angstadt and Calabrese, 1989; Golowasch and Marder, 1992). Depolarizing current pulses also revealed time-dependent voltage responses in B8. When stepped to levels more depolarized than –35 mV, the membrane potential response exhibited a gradual depolarizing drift (Fig. 4B1).
In view of the time-dependent properties of the voltage deflections in B8, two values were measured for each current pulse. When initial values of voltage responses (designated "early" in Fig. 4B2, closed symbols) were plotted, a linear relation comparable to that observed in B16 was obtained. However, voltage values measured immediately prior to the termination of current pulses (designated "late" in Fig. 4B2, open symbols) revealed departures from ohmic behavior in both the hyperpolarizing and depolarizing quadrants. These deviations reflect, respectively, the sag and the drift described above.
Longer current pulses (5 s) were delivered to B8 to further examine the slowly developing response to depolarization. With increasing pulse amplitudes, the time constant of the onset of voltage responses was unchanged (indicated at the left of each voltage trace of Fig. 4C). The time constant of decay, however, was progressively increased (indicated at the right of each record), suggesting that the depolarizing drift in B8 is associated with an increase in the membrane resistance. The gradual depolarizations produced by injection of larger current pulses could exceed threshold and elicit repetitive impulses (lowest records).
The possible influence of the membrane properties of B16 and B8 on EPSPs was examined using brief depolarizing pulses as a crude simulation of synaptic currents (Fig. 5). The amplitude and duration of a current pulse were adjusted to produce a depolarizing response that resembled a B20-evoked EPSP in B16. At the resting membrane potential of B16 (–48 mV), this pulse produced a depolarization of 2 mV with a half-time of decay of 80 ms (Fig. 5A1, black recording). When the membrane was hyperpolarized to a level 40 mV more hyperpolarized than rest, the amplitude of the response was increased, but its rate of decay was unchanged (Fig. 5A1, gray recording). In contrast, a similar manipulation of the membrane potential in B8 produced a marked increase in the decay kinetics of its response to a pulse of 4 nA. Whereas responses at rest (–46 mV) exhibited a decay half time of 85 ms (Fig. 5B1, black recording), those elicited with the Vm set 40 mV more hyperpolarized than rest decayed with a half time of 35 ms (Fig. 5B1, gray recording). The amplitude of the voltage response in B8 was relatively unaffected by hyperpolarization (see Discussion).
The differential effects of membrane potential on responses to current pulses suggested that the specific membrane properties of B16 and B8 could contribute to the observed differences in the summation of their EPSPs originating from B20. This possibility was examined by injecting trains of pulses to simulate the EPSP trains that occur during B20 bursts. Whereas the membrane potential of B16 decayed to rest (–52 mV) between each pulse (Fig. 5A2, upper trace), those in B8 exhibited summation when the membrane was initially at its resting level (–49 mV; Fig. 5B2, upper trace). When the membrane potential of B8 was pre-set to –89 mV, each pulse returned to this level prior to the onset of the subsequent response (Fig. 5B2, lower trace). These findings are consistent with the observed dependence of synaptic summation on the membrane potential of B8 (Fig. 2B, D).
Together, these results indicate that the divergent signaling from B20 to two motor neurons, B16 and B8, exhibits distinctions in two forms of use-dependent increases in efficacy, facilitation, and summation. Although the differences in facilitation appear to reflect target-specific properties of plasticity at B20 terminals, the difference in summation can be attributed to dissimilar membrane properties in the two postsynaptic cells. The facilitation and summation both bias the excitatory signaling from B20 toward B8 versus its signaling to B16.
GABAergic regulation of divergent synaptic enhancement from B20 to B16 and B8
GABA-like immunoreactivity is present in B20 (Díaz-Ríos et al., 2002), and GABA has been shown to modify synaptic signaling of this neuron via GABAB-like receptors (Díaz-Ríos and Miller, 2005). It was therefore of interest to examine the effects of GABA on facilitation and summation of B20-evoked EPSPs in B16 and B8. Application of exogenous GABA (up to 1 mM) did not affect facilitation or summation of the excitatory signaling of B20 to B16 (Fig. 6A1,2). Facilitation of EPSP10 in the presence of GABA (0.48 ± 0.14; mean ± SEM) did not differ from control values (0.37 ± 0.16; t = 1.22; P > 0.05; n = 4; Fig. 6B1). Likewise, the level of summation of EPSP10 in GABA (2.1 ± 0.2 mV) did not differ from control values (2.4 ± 0.3 mV; t = 0.83; P > 0.05; n = 4; Fig. 6B2).
In contrast, application of GABA had potent effects on both facilitation and summation of rapid signaling from B20 to B8 (Fig. 7A1,2). Facilitation of EPSP10 in the presence of GABA (3.7 ± .8; mean ± SEM) was significantly greater than control values (2.6 ± 0.3; t = 1.4; P < 0.05; n = 5; Fig. 7B1). Likewise, the level of summation of EPSP10 in GABA (4.2 ± 0.5 mV) was significantly greater than control values (2.9 ± 0.2 mV; t = 2.69; P < 0.05; n = 5; Fig. 7B2). Each of the actions observed with GABA was also produced by the GABAB agonist baclofen (Fig. 8A1,2). Facilitation of EPSP10 in the presence of baclofen (1.6 ± 0.2) was significantly greater than control values (0.6 ± 0.1; t = 4.43; P < 0.05; n = 5; Fig. 8B1). The level of summation of EPSP10 in baclofen (7.9 ± 0.4 mV) was also significantly greater than control values (3.3 ± 0.1 mV; t = 7.60; P < 0.05; n = 5; Fig. 8B2).
When B8 was at its resting potential, it was not possible to examine the effects of GABA on synaptic facilitation or summation individually. But adjusting the membrane potential of B8 to levels 40 mV more hyperpolarized than rest markedly diminished summation of the B20-to-B8 EPSPs (see Fig. 3), enabling a more direct examination of GABAergic effects on facilitation of this signal (Fig. 9). GABA (1 mM) produced a significant increase (t = 6.89; P < 0.05) in the facilitation value of EPSP10 which was reversed upon wash with normal ASW (Fig. 9A, C1). Baclofen (1 mM) produced a comparable increase (t = 5.95; P < .05) in the facilitation of EPSP10 in B8 when it was pre-set to hyperpolarized membrane potentials (Fig. 9B, C2). The increases in summation that were elicited by GABA and baclofen when B8 was at its resting potential (Fig. 7 and Fig. 8) were not observed at hyperpolarized levels.
Figure 9. GABA and baclofen enhance facilitation of the B20-to-B8 EPSP. (A1) B8 was hyperpolarized via current injection to –89 mV prior to eliciting a train of impulses in B20. (A2) Facilitation of the B20-to-B8 EPSP was increased in the presence of GABA (1 mM). (A3) The GABA-induced increase in facilitation was reversed by washing with normal ASW. (B1) B8 was hyperpolarized via current injection to –103 mV prior to eliciting a train of impulses in B20. (B2) Facilitation of the B20-to-B8 EPSP was increased in the presence of baclofen (1 mM). (B3) The baclofen-induced increase in facilitation was reversed by washing with normal ASW. (C1) Grouped data (n = 5) show that facilitation of EPSP10 was significantly enhanced by GABA when B8 was hyperpolarized 40 mV more hyperpolarized than rest prior to eliciting a B20 impulse train. (C2) Grouped data (n = 5) show that facilitation of EPSP10 was significantly enhanced by baclofen when B8 was 40 mV more hyperpolarized than rest prior to eliciting a B20 impulse train.
Finally, the effects of baclofen on summation were examined using small current pulses (250 pA, 20 ms) injected into B8 to simulate synaptic currents without firing the presynaptic neuron B20 (Fig. 10). It was previously shown that trains of such pulses can exhibit "summation" when applied within the physiological firing frequency of B20 (Fig. 5). Application of baclofen (1 mM) decreased the rate of decay of voltage deflections such that successive pulses within a train were incrementally prolonged and their summed depolarization of B8 was increased (Fig. 10B–D). As the effect of baclofen developed, the cumulative depolarization of pulse trains produced firing of B8 at progressively earlier phases (Fig. 10C, D).
Figure 10. Baclofen increases the decay time of voltage deflections produced by current pulses in B8. (A) A train of current pulses (5 ms, 0.25 nA) was injected into B8 to mimic the EPSPs that are produced by a burst of B20 impulses. (B–D) In the presence of baclofen (1 mM), the decay times of the voltage deflections became successively prolonged. Pulse trains that failed to reach the threshold of B8 under control conditions became capable of producing action potentials at progressively earlier phases of their duration.
Together, these results demonstrate that GABA, acting via GABAB-like receptors, enhances facilitation and summation from B20 to B8 but not to B16. The increase in B20-to-B8 facilitation appears to reflect presynaptic properties that are independent of the postsynaptic membrane potential. The increase in summation can be attributed to postsynaptic actions in B8 that produce decreased rates of decay in successive EPSPs.
Discussion
In this study, signaling from the influential buccal interneuron B20 was progressively biased toward one of its followers during trains of action potentials. Our observations indicate that this bias resulted from presynaptic and postsynaptic mechanisms acting in a concerted fashion (Fig. 11A). Both contributions to this bias were amplified by modulatory effects of GABA, which increased facilitation and summation in a target-specific fashion (11B). These findings predict that, excluding additional factors, patterned firing of B20 will be more effective in producing excitation of B8 than of B16. This was in fact observed with simultaneous recording of the two motor neurons when trains of impulses were produced in B20 (Fig. 11C).
Figure 11. Divergent short-term synaptic plasticity and modulatory metaplasticity in the rapid excitatory signaling from B20 to B16 and B8. (A) Schematic of divergent excitatory signaling from B20 to the two radula closer motor neurons B16 and B8. Short-term facilitation and summation were greater in the rapid EPSPs to B8 (labeled with larger type). Differences in facilitation are attributed to target-specific properties of the B20 synapses. Differences in summation are attributed to dissimilar membrane properties of the two postsynaptic motor neurons. (B) Proposed modulatory effects of GABA on the divergent signaling of B20. Actions of GABA were not detected on either facilitation or summation of the EPSPs from B20 to B16. In B8, both of these short-term increases in the synaptic efficacy of signaling of from B20 were enhanced by GABA and baclofen. GABAB effects on facilitation are proposed to reflect actions on the B20-to-B8 presynaptic terminals. Effects on summation are proposed to reflect GABAB-like actions on the membrane properties of B8. (C) When repetitive trains of B20 impulses were produced by injection of current pulses (bottom record), facilitation and summation of EPSPs contributed to firing B8 while responses in B16 remained subthreshold. Recording of the impulse in B8 was truncated by the data acquisition system.
Previous investigations localized markers for catecholamines and GABA to B20 (Díaz-Ríos et al., 2002) and showed that its fast EPSPs to both B16 and B8 were mediated by dopamine (Díaz-Ríos and Miller, 2005). While it is intriguing to consider that GABA could be acting as a cotransmitter at these synapses, its release from B20 remains to be demonstrated, and its source is therefore not specified in our present schema (Fig. 11B). However, whether the modulation of signaling from B20 is homosynaptic or heterosynaptic in origin, its presence in an interneuron that is embedded within the buccal CPG would classify it as an intrinsic form of modulation (Katz et al., 1994; Katz and Frost, 1996; see Introduction). As such, its magnitude can be expected to both reflect and influence the degree to which the overall system is activated.
A type of modulation with formal similarities to that observed in this study has been demonstrated in the circuit mediating the siphon withdrawal reflex of Aplysia (Fischer et al., 1997). In that system, serotonin acting in a heterosynaptic fashion attenuated the ability of an identified inhibitory neuron (L30) to display short-term synaptic enhancement. This action was designated "modulatory metaplasticity" to distinguish it from previously described homosynaptic forms of metaplasticity in which a neuron’s firing history influenced its ability to express plasticity (Abraham and Bear, 1996). If GABA is released when B20 fires, and if that synaptically released GABA is capable of influencing subsequent rapid dopaminergic signaling, then both of these terms would be applicable to B20-to-B8 signaling during impulse trains. In the context of the existing nomenclature, such regulation would be designated "homosynaptic modulatory metaplasticity" (Fig. 11B).
Divergent synaptic plasticity
Branch, or target-specific, synaptic plasticity, such as that observed in the signaling from B20 to its two follower motor neurons, appears to be used extensively to achieve divergent channeling of information flow in motor systems. In the neuromuscular systems of crustaceans, where a small number of neurons control multiple muscles, terminals of an individual motor neuron can exhibit wide ranges of facilitation or depression (Atwood, 1967; Bittner, 1968). In an extreme case, the terminals of a single stomatogastric motor neuron in the lobster were shown to exhibit facilitation in one muscle and depression in another muscle (Katz et al., 1993).
Within the Aplysia buccal ganglion, diverse manifestations of differential synaptic plasticity can contribute to CPG function. Gardner and Kandel (1977) identified a diphasic synaptic potential (EPSP followed by an IPSP) originating from the cholinergic interneuron B4/5. Repeated firing of B4/5 preferentially reduced the magnitude of the hyperpolarizing component of this PSP, eventually resulting in a monophasic depolarization. In that instance, differential rates of synaptic depression were attributed to properties of distinct postsynaptic acetylcholine receptors that exhibited different rates of desensitization (Gardner and Kandel, 1977). Another form of differential synaptic plasticity was recently shown to occur in the signaling of buccal interneuron B65, where EPSPs exhibit depression in one follower neuron (B4/5) and facilitation in a second (B8; Kabotyanski et al., 1998). Interestingly, B65 shares the GABA-DA neurotransmitter phenotype with B20, the subject of this investigation (Díaz-Ríos et al., 2002; Due et al., 2004).
Complementary pre- and postsynaptic modulation of plasticity
Previously, it was found that GABA, acting via GABAB-like receptors, produced a small depolarization and increase in the input resistance and excitability of B8 (Díaz-Ríos and Miller, 2005). GABA and baclofen were also found to increase the amplitude of EPSPs evoked in B8 by single impulses in B20. The present investigation demonstrated that GABA can also enhance signaling from B20 to B8 by increasing facilitation and summation. Additional mechanisms, including postsynaptic interactions between the neurotransmitters present in B20, may also be operating to modify these signals. Recently, it was shown that GABA and baclofen increase inward currents produced by dopamine in B8 (Svensson et al., 2004). If GABA is in fact co-released with dopamine from B20, then such synergistic influences could contribute to the enhancement of the B20-to-B8 EPSP that is observed during impulse trains.
Presynaptic modulation.
GABAB receptors have been shown to regulate transmitter release in a range of systems and species (see review by Bettler et al., 2004), including several invertebrates (Miwa et al., 1990; Parnas et al., 1999; Gutovitz et al., 2001). Prior to the recognition of GABA receptor subtypes, Phillipe and coworkers (1981) found that baclofen (3–5 x 10–5 M) altered synaptic plasticities in the Aplysia abdominal ganglion. In that study, the effects of baclofen were thought to be indirect and were attributed to its ability to liberate biogenic amines (Philippe et al., 1981). In the system studied in the present investigation, the concentrations of GABA and baclofen required to produce detectable effects were considerably higher than are normally found to activate GABAB receptors. However, if GABA originates from the terminals of B20 itself, it is likely to be capable of achieving high concentrations at or near the sites of dopamine release.
We propose that the GABA-induced increases in facilitation reflect presynaptic actions that modify the history-dependence of neurotransmitter release from B20. Similar presynaptic modulatory actions have been demonstrated with aminergic neuromodulators in Aplysia central synapses (Tremblay et al., 1976; Woodson et al., 1976; Newlin et al., 1980). Their possible behavioral significance is best understood in the circuits that mediate defensive reflexes, where presynaptic modulation is implemented by serotonin acting in a heterosynaptic fashion (Carew and Kandel, 1974; Kandel and Schwartz, 1982; Byrne and Kandel, 1996).
Postsynaptic modulation.
Our findings indicate that GABA, acting via GABAB-like receptors on B8, enhances summation of the B20-to-B8 EPSP. This modulatory action did not occur when the membrane potential of B8 was set to hyperpolarized levels. GABA did, however, enhance the "summation" of voltage deflections produced by current pulses injected into B8 at its resting Vm. Its effects appear, therefore, to reflect an action on membrane properties of B8 that are operative near its resting membrane potential. We propose that the progressive advance of the B8 Vm into a region of increased resistance during a train of EPSPs contributes to the enhanced summation of successive EPSPs and that the modulatory effects of GABA reflect an action that augments this intrinsic property.
Several buccal interneurons and sensory neurons converge upon B8 and B16, and their synaptic actions on these motor neurons have been related to their ability to specify motor programs (Kabotyanski et al., 1998; Nargeot et al., 1999b; Jing and Weiss, 2001; Morgan et al., 2002; Jing et al., 2003; Proekt et al., 2004). A previous investigation demonstrated a nonlinearity in the membrane properties of B8 that could modify EPSPs originating from the identified mechanosensory neuron B21 (Klein et al., 2000). In that study, the amplitude and time constant of decay of the B21-to-B8 EPSP were both decreased when the membrane potential of B8 was set to hyperpolarized levels. The nonlinear properties of B8 were likened to the "anomalous rectification" that occurs in the serotonergic metacerebral cell (MCC) of Aplysia (Kandel and Tauc, 1966). In the MCC, anomalous rectification strongly influenced the ability of repetitive EPSPs to summate (Kandel and Tauc, 1966). In agreement with observations made in the MCC, we found that the effect of the B8 membrane potential on the rate of decay of the B20-to-B8 EPSP was substantially greater than its effect on the magnitude of this EPSP.
Divergent modulation of plasticity—potential contributions to behavioral plasticity
The phase of radula closure with respect to its protraction and retraction is the operational parameter that distinguishes ingestive from egestive feeding behaviors (Kupfermann, 1974; Morton and Chiel, 1993a, Morton and Chiel, 1993b; Cropper et al., 2004). In experiments with both in vivo and in vitro preparations, radula closure is commonly monitored by observing the phase and pattern of B8 firing (Church and Lloyd, 1994; Nargeot et al., 1997, Nargeot et al., 1999a, Nargeot et al., 1999b; Kabotyanski et al., 1998; Sweedler et al., 2002). The influence of B20 on the buccal CPG is best understood in the context of egestive motor patterns produced by stimulation of cerebral-buccal command neurons (Jing and Weiss, 2001) or the esophageal nerve (Proekt et al., 2004). In both instances, the direct B20-to-B8 EPSP is thought to specify egestive motor programs by promoting radula closure during protraction. The degree to which the forms of synaptic enhancement examined in this study contribute to such motor-pattern specification remains uncertain. Several observations suggest that additional more persistent forms of plasticity also modify the signaling of B20 to B8 (Teyke et al., 1993; Jing and Weiss, 2001; Proekt et al., 2004; our observations).
The initiation of a feeding bout in Aplysia is characterized by a progressive increase in the frequency and intensity of biting, a feature that is thought to reflect food-induced arousal (Kupfermannn et al., 1979; Rosen et al., 1989). Transitions of buccal motor programs also occur in a graded fashion and exhibit some inertia (Kabotyanski et al., 1998; Brezina et al., 2003a, Brezina et al., 2003b; Proekt et al., 2004). A progressive increase in the efficacy of excitatory signaling from B20 to B8 was recently shown to accomplish the graded transition of the buccal CPG toward egestive motor patterns (Proekt et al., 2004). Motor-pattern warm-up and transitions are likely to require that radula closer motor neurons be recruited in a specific sequence (Brezina et al., 2003a, Brezina et al., 2003b; Zhurov et al., 2005; Ye et al., 2006). The divergent short-term synaptic plasticities from B20 to B16 and B8, and their differential modulation by GABA, could contribute to the effective and adaptive onset and transitioning of repetitive motor patterns.
Recent investigations have increased our appreciation for the scope and complexity of neural mechanisms that contribute to relatively simple forms of behavioral plasticity in Aplysia (Glanzman, 1995; Byrne and Kandel, 1996; Martin et al., 2000). The mechanisms underlying learning and memory in the polymorphic circuits that produce repetitive behaviors are likely to exhibit comparable or even greater levels of distribution and complexity (see Benjamin et al., 2000). Correlates of associative conditioning in molluscan feeding circuits have been shown to occur in the sensory pathways that mediate food detection (Morielli et al., 1986; Staras et al., 1999; Lechner et al., 2000a; Mozzachiodi et al., 2003), in the signaling from command-like interneurons to pattern-initiating CPG elements (Kovac et al., 1986; Lechner et al., 2000b; Straub et al., 2004), and in the biophysical properties of key buccal interneurons (Nargeot et al., 1999a, Nargeot et al., 1999b; Brembs et al., 2002). The contribution of plastic synaptic signaling to the motor neurons themselves, a major determinant of non-CPG-mediated behavioral plasticity (see contributions to this symposium), has received less scrutiny. This report aims to stimulate further investigation into modifications of signaling to the final common pathway that executes such CPG-mediated motor programs when the experience of an animal causes it to adjust its actions.
Acknowledgments
Supported by the National Science Foundation: CAREER Award: IBN-9722349; National Institutes of Health DRS RCMI RR-03051, NIGMS MBRS: GM-08224, and NIMH MRISP MH48190; and funds from the University of Puerto Rico Medical Sciences Campus Deanship of Biomedical Sciences.
Footnotes
Received 7 November 2005; accepted 23 January 2006.
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Abstract
The contributions to this symposium are unified by their focus on the role of synaptic plasticity in sensorimotor learning. Synaptic plasticities are also known to operate within the central pattern generator (CPG) circuits that produce repetitive motor programs, where their relation to adaptive behavior is less well understood. This study examined divergent synaptic plasticity in the signaling of an influential interneuron, B20, located within the CPG that controls consummatory feeding-related behaviors in Aplysia. Previously, B20 was shown to contain markers for catecholamines and GABA (Díaz-Ríos et al., 2002), and its rapid synaptic signaling to two follower motor neurons, B16 and B8, was found to be mediated by dopamine (Díaz-Ríos and Miller, 2005). In this investigation, two incremental forms of increased synaptic efficacy, facilitation and summation, were both greater in the signaling from B20 to B8 than in the signaling from B20 to B16. Manipulation of the membrane potentials of the two postsynaptic motor neurons did not affect facilitation of excitatory postsynaptic potentials (EPSPs) to either follower cell. Striking levels of summation in B8, however, were eliminated at hyperpolarized membrane potentials and could be attributed to distinctive membrane properties of this postsynaptic cell. GABA and the GABAB agonist baclofen increased facilitation and summation of EPSPs from B20 to B8, but not to B16. The enhanced facilitation was not affected when the membrane potential of B8 was pre-set to hyperpolarized levels, but GABAergic effects on summation were eliminated by this manipulation. These observations demonstrate a target-specific amplification of synaptic efficacy that can contribute to channeling the flow of divergent information from an intrinsic interneuron within the buccal CPG. They further suggest that GABA, acting as a cotransmitter in B20, could induce coordinated and target-specific pre- and postsynaptic modulation of these signals. Finally, we speculate that target-specific plasticity and its modulation could be efficient, specific, and flexible substrates for learning-related modifications of CPG function.
Introduction
The contributions to this symposium underscore the central position of activity-dependent synaptic plasticity in our present understanding of sensorimotor learning. One attribute of such plasticities that renders them suitable for producing adaptive behavior is their own capacity to be modified (Krasne, 1978; Byrne and Kandel, 1996; Abraham and Bear, 1996). Recent investigations have broadened our appreciation for the occurrence of diverse forms of synaptic plasticity within the central pattern generator (CPG) circuits that produce repetitive behaviors (Marder, 1998; Parker and Grillner, 1999, Parker and Grillner, 2000; Nadim and Manor, 2000; Sakurai and Katz, 2003; Yuste et al., 2005). The contribution of such activity-dependent modifications of synaptic strength to adaptive CPG-driven motor activity, however, remains incompletely understood.
The consummatory behaviors of Aplysia are generated by a multifunctional or polymorphic CPG that supports several forms of non-associative and associative behavioral plasticity (Susswein and Schwarz, 1983; Kupfermann et al., 1989; Colwill et al., 1997; Lechner et al., 2000a). Cellular analyses of in vitro analogs have begun to reveal potential correlates of experience-dependent plasticity at multiple levels of the feeding network (Nargeot et al., 1997, Nargeot et al., 1999a, Nargeot et al., 1999b; Lechner et al., 2000b; Brembs et al., 2002, Brembs et al., 2004; Mozzachiodi et al., 2003; Proekt et al., 2004). These correlates often correspond to loci where activity-dependent synaptic plasticity is known to occur. For example, command-like interganglionic projection neurons in the cerebral ganglion activate CPG circuits in the buccal ganglion via excitatory postsynaptic potentials (EPSPs) that exhibit various forms of short-term enhancement (Sánchez and Kirk, 2000, Sánchez and Kirk, 2002; Hurwitz et al., 2003). Signaling from the interneurons within the buccal CPG to the motor neurons that execute consummatory feeding behaviors also displays several forms of plasticity (Teyke et al., 1993; Kabotyanski et al., 1998; Jing and Weiss, 2002). Finally, patterned firing by the motor neurons themselves produce facilitating or depressing excitatory junction potentials in the buccal muscles (Cohen et al., 1978; Cropper et al., 1990; Jordan et al., 1993). The possible involvement of these synaptic plasticities with feeding-related behavioral plasticities is largely unexplored.
Regulation of synaptic efficacy by neuromodulatory messengers also plays a pivotal role in learning and memory in several invertebrate model systems (Kandel and Schwartz, 1982; Hawkins et al., 1993; Menzel, 2001; Balaban, 2002). Neuromodulatory control has been intensively studied in CPG circuits, where broadly acting messengers are capable of regulating the biophysical properties of constituent neurons and their synaptic connections in a coherent fashion (Kupfermann, 1979; Harris-Warrick and Marder, 1991; Katz, 1999). When modulators originate from neurons that are not sensu stricto participants in the CPG, they are considered extrinsic (Kupfermann et al., 1979; Morgan et al., 2000). When they derive from neurons that are themselves elements of the CPG (Katz and Frost, 1996), or from motor neurons (Cropper et al., 1987), they are designated intrinsic. In both configurations, modulators are frequently released from neurons in which they occur as cotransmitters (Kupfermann, 1991; Weiss et al., 1992; Nusbaum et al., 2001).
Recently, colocalization of markers for GABA and catecholamines was demonstrated in B20 (Díaz-Ríos et al., 2002), an intrinsic buccal CPG interneuron that can initiate and specify the consummatory feeding motor programs of Aplysia (Teyke et al., 1993; Jing and Weiss, 2001; Proekt et al., 2004). Pharmacological evidence supported the role of dopamine as the mediator of fast synaptic signaling from B20 to B16 and B8, two motor neurons that cause closure of the food-grasping radula (Fig. 1A; see Díaz-Ríos and Miller, 2005). GABA, acting via GABAB-like receptors, exerted differential modulatory actions on synaptic transmission from B20 to B16 and B8 (Díaz-Ríos and Miller, 2005). In that study, the effects of GABA were tested on EPSPs that were evoked by single B20 impulses. As B20 ordinarily fires in a burst mode during feeding motor programs (Teyke et al., 1993; Jing and Weiss, 2001; Proekt et al., 2004), this investigation explored some plastic properties of its divergent synaptic signaling and their modification by GABA. Some of these observations have appeared in abstract form (Díaz-Ríos and Miller, 2002).
Materials and Methods
Subjects
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, Miami, FL) or from Marinus Inc. (Long Beach, CA). Animals were maintained in a refrigerated aquarium (14–16 °C) and fed dried seaweed twice per week. Experimental protocols followed established guidelines of the Institutional Animal Care and Use Committee of the University of Puerto Rico Medical Sciences Campus.
Electrophysiology and pharmacology
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 to record from B20 and its direct synaptic follower motor neurons B16 and B8 (Teyke et al., 1993). Current was injected into the postsynaptic cells by a second intracellular electrode (5–10 M). B20 was stimulated via passage of current through the recording electrode across the bridge circuit of the amplifier (NeuroProbe, A-M Systems). All experiments were conducted in artificial seawater (ASW) containing high concentrations of divalent cations (Liao and Walters, 2002) to attenuate polysynaptic activity.
Solutions of drugs at the concentration to be applied were prepared in high-divalent ASW immediately before the start of each experiment. GABA and (±)-baclofen were obtained from Sigma Chemical Co. (St. Louis, MO). All experiments were performed with application of agonists at a concentration of 1 mM, which produced consistent and reversible effects in previous studies (see Díaz-Ríos and Miller, 2005). Drugs were delivered at a rate of 0.5 ml/min by a gravity-fed perfusion system (ALA Scientific Instruments, model VM4). Responses were recorded 2, 5, and 10 min after switching the perfusion source.
Measurements and statistics
A measurement of summation (Sumn; unit: mV) for a particular EPSP in a train (EPSPn) was obtained by subtracting the postsynaptic membrane potential prior to initiation of the train (Vrest) from the membrane potential immediately prior to the onset of EPSPn (Fig. 1B). An operational measure of facilitation of EPSPn (Fn, a unitless value) was defined as (EPSPn/EPSP1) – 1, so that a value of 0 signified the absence of facilitation (Fig. 1B). EPSP amplitudes were corrected for summation by subtracting Sumn. They were also corrected for changes in driving force (Martin, 1955), using an estimated value of +5 mV as the reversal potential of B20-evoked fast EPSPs (Díaz-Ríos and Miller, 2005).
In view of the presence of long-lasting forms of plasticity and GABAergic actions in this system (Díaz-Ríos and Miller 2002, Díaz-Ríos and Miller 2005), statistical tests (Student’s paired t test; two-tailed) were performed by comparing measurements obtained prior to drug application to measurements obtained at the peak of the response. A value of P < 0.05 was established as the criterion for significance.
Results
Divergent synaptic signaling from B20 to B16 and B8
In a previous study, we found the rapid synaptic signaling from B20 to B16 and B8 to be mediated by dopamine (See Fig. 1A andDíaz-Ríos and Miller, 2005). During that investigation, repetitive firing of B20 revealed two forms of incremental increases in the efficacy of its excitatory signaling—facilitation and summation (Fig. 1B; see also Teyke et al., 1993; Jing and Weiss, 2001). Simultaneous recording from B16 and B8 allowed us to directly compare facilitation and summation during their responses to B20 impulse trains (Fig. 2A). Facilitation values (see definition in Materials and Methods) measured at the 10th EPSP in a train in B8 (1.8 ± 0.4; mean ± SEM) were greater than those observed in B16 (1.2 ± 0.2; t = 4.63; P < 0.05; n = 4; Fig. 2B). Summation values (see definition in Materials and Methods) measured at the tenth EPSP in a train in B8 (6.1 ± 0.5 mV) were also greater than those observed in B16 (4.3 ± 0.3 mV; t = 7.59, P < 0.05; n = 4; Fig. 2C).
The activity-dependent short-term plasticity of EPSPs originating from B20 was further examined in experiments in which the postsynaptic membrane potentials of B16 and B8 were set to hyperpolarized levels. Synaptic facilitation, which is generally thought to reflect presynaptic conditions that produce increases in neurotransmitter release (del Castillo and Katz, 1954; Fisher et al., 1997; Zucker and Regher, 2002), was not affected by this manipulation (Fig. 2A–C). Summation, on the other hand, was differentially affected in the two synaptic followers. Whereas the measures of summation increased in B16 when that cell was pre-set to hyperpolarized levels (Fig. 3A, D), summation was severely attenuated in B8 when it was hyperpolarized (Fig. 3B, D).
The differential effects of membrane potential on synaptic summation in B16 and B8 prompted us to compare the current-voltage relations in these two motor neurons. Voltage responses to current pulses (2 s) injected into B16 rapidly reached a stable value (Fig. 4A). A plot of these voltage displacements as a function of injected current values revealed a predominantly linear relation with limited rectification in the depolarizing quadrant (Fig. 4A2). Responses to current pulses in B8 were more complex (see also Klein et al., 2000; Díaz-Ríos and Miller, 2005). Pulses to levels more hyperpolarized than about –70 mV exhibited a depolarizing sag (Fig. 4B1, arrow). Upon release from these large pulses, the membrane potential of B8 displayed a transient depolarization (Fig. 4B1, arrowhead) prior to reestablishing its resting level. Although not further explored here, such responses have been shown to reflect the presence of hyperpolarization-activated inward currents (Ih) in other invertebrate motor systems (Angstadt and Calabrese, 1989; Golowasch and Marder, 1992). Depolarizing current pulses also revealed time-dependent voltage responses in B8. When stepped to levels more depolarized than –35 mV, the membrane potential response exhibited a gradual depolarizing drift (Fig. 4B1).
In view of the time-dependent properties of the voltage deflections in B8, two values were measured for each current pulse. When initial values of voltage responses (designated "early" in Fig. 4B2, closed symbols) were plotted, a linear relation comparable to that observed in B16 was obtained. However, voltage values measured immediately prior to the termination of current pulses (designated "late" in Fig. 4B2, open symbols) revealed departures from ohmic behavior in both the hyperpolarizing and depolarizing quadrants. These deviations reflect, respectively, the sag and the drift described above.
Longer current pulses (5 s) were delivered to B8 to further examine the slowly developing response to depolarization. With increasing pulse amplitudes, the time constant of the onset of voltage responses was unchanged (indicated at the left of each voltage trace of Fig. 4C). The time constant of decay, however, was progressively increased (indicated at the right of each record), suggesting that the depolarizing drift in B8 is associated with an increase in the membrane resistance. The gradual depolarizations produced by injection of larger current pulses could exceed threshold and elicit repetitive impulses (lowest records).
The possible influence of the membrane properties of B16 and B8 on EPSPs was examined using brief depolarizing pulses as a crude simulation of synaptic currents (Fig. 5). The amplitude and duration of a current pulse were adjusted to produce a depolarizing response that resembled a B20-evoked EPSP in B16. At the resting membrane potential of B16 (–48 mV), this pulse produced a depolarization of 2 mV with a half-time of decay of 80 ms (Fig. 5A1, black recording). When the membrane was hyperpolarized to a level 40 mV more hyperpolarized than rest, the amplitude of the response was increased, but its rate of decay was unchanged (Fig. 5A1, gray recording). In contrast, a similar manipulation of the membrane potential in B8 produced a marked increase in the decay kinetics of its response to a pulse of 4 nA. Whereas responses at rest (–46 mV) exhibited a decay half time of 85 ms (Fig. 5B1, black recording), those elicited with the Vm set 40 mV more hyperpolarized than rest decayed with a half time of 35 ms (Fig. 5B1, gray recording). The amplitude of the voltage response in B8 was relatively unaffected by hyperpolarization (see Discussion).
The differential effects of membrane potential on responses to current pulses suggested that the specific membrane properties of B16 and B8 could contribute to the observed differences in the summation of their EPSPs originating from B20. This possibility was examined by injecting trains of pulses to simulate the EPSP trains that occur during B20 bursts. Whereas the membrane potential of B16 decayed to rest (–52 mV) between each pulse (Fig. 5A2, upper trace), those in B8 exhibited summation when the membrane was initially at its resting level (–49 mV; Fig. 5B2, upper trace). When the membrane potential of B8 was pre-set to –89 mV, each pulse returned to this level prior to the onset of the subsequent response (Fig. 5B2, lower trace). These findings are consistent with the observed dependence of synaptic summation on the membrane potential of B8 (Fig. 2B, D).
Together, these results indicate that the divergent signaling from B20 to two motor neurons, B16 and B8, exhibits distinctions in two forms of use-dependent increases in efficacy, facilitation, and summation. Although the differences in facilitation appear to reflect target-specific properties of plasticity at B20 terminals, the difference in summation can be attributed to dissimilar membrane properties in the two postsynaptic cells. The facilitation and summation both bias the excitatory signaling from B20 toward B8 versus its signaling to B16.
GABAergic regulation of divergent synaptic enhancement from B20 to B16 and B8
GABA-like immunoreactivity is present in B20 (Díaz-Ríos et al., 2002), and GABA has been shown to modify synaptic signaling of this neuron via GABAB-like receptors (Díaz-Ríos and Miller, 2005). It was therefore of interest to examine the effects of GABA on facilitation and summation of B20-evoked EPSPs in B16 and B8. Application of exogenous GABA (up to 1 mM) did not affect facilitation or summation of the excitatory signaling of B20 to B16 (Fig. 6A1,2). Facilitation of EPSP10 in the presence of GABA (0.48 ± 0.14; mean ± SEM) did not differ from control values (0.37 ± 0.16; t = 1.22; P > 0.05; n = 4; Fig. 6B1). Likewise, the level of summation of EPSP10 in GABA (2.1 ± 0.2 mV) did not differ from control values (2.4 ± 0.3 mV; t = 0.83; P > 0.05; n = 4; Fig. 6B2).
In contrast, application of GABA had potent effects on both facilitation and summation of rapid signaling from B20 to B8 (Fig. 7A1,2). Facilitation of EPSP10 in the presence of GABA (3.7 ± .8; mean ± SEM) was significantly greater than control values (2.6 ± 0.3; t = 1.4; P < 0.05; n = 5; Fig. 7B1). Likewise, the level of summation of EPSP10 in GABA (4.2 ± 0.5 mV) was significantly greater than control values (2.9 ± 0.2 mV; t = 2.69; P < 0.05; n = 5; Fig. 7B2). Each of the actions observed with GABA was also produced by the GABAB agonist baclofen (Fig. 8A1,2). Facilitation of EPSP10 in the presence of baclofen (1.6 ± 0.2) was significantly greater than control values (0.6 ± 0.1; t = 4.43; P < 0.05; n = 5; Fig. 8B1). The level of summation of EPSP10 in baclofen (7.9 ± 0.4 mV) was also significantly greater than control values (3.3 ± 0.1 mV; t = 7.60; P < 0.05; n = 5; Fig. 8B2).
When B8 was at its resting potential, it was not possible to examine the effects of GABA on synaptic facilitation or summation individually. But adjusting the membrane potential of B8 to levels 40 mV more hyperpolarized than rest markedly diminished summation of the B20-to-B8 EPSPs (see Fig. 3), enabling a more direct examination of GABAergic effects on facilitation of this signal (Fig. 9). GABA (1 mM) produced a significant increase (t = 6.89; P < 0.05) in the facilitation value of EPSP10 which was reversed upon wash with normal ASW (Fig. 9A, C1). Baclofen (1 mM) produced a comparable increase (t = 5.95; P < .05) in the facilitation of EPSP10 in B8 when it was pre-set to hyperpolarized membrane potentials (Fig. 9B, C2). The increases in summation that were elicited by GABA and baclofen when B8 was at its resting potential (Fig. 7 and Fig. 8) were not observed at hyperpolarized levels.
Figure 9. GABA and baclofen enhance facilitation of the B20-to-B8 EPSP. (A1) B8 was hyperpolarized via current injection to –89 mV prior to eliciting a train of impulses in B20. (A2) Facilitation of the B20-to-B8 EPSP was increased in the presence of GABA (1 mM). (A3) The GABA-induced increase in facilitation was reversed by washing with normal ASW. (B1) B8 was hyperpolarized via current injection to –103 mV prior to eliciting a train of impulses in B20. (B2) Facilitation of the B20-to-B8 EPSP was increased in the presence of baclofen (1 mM). (B3) The baclofen-induced increase in facilitation was reversed by washing with normal ASW. (C1) Grouped data (n = 5) show that facilitation of EPSP10 was significantly enhanced by GABA when B8 was hyperpolarized 40 mV more hyperpolarized than rest prior to eliciting a B20 impulse train. (C2) Grouped data (n = 5) show that facilitation of EPSP10 was significantly enhanced by baclofen when B8 was 40 mV more hyperpolarized than rest prior to eliciting a B20 impulse train.
Finally, the effects of baclofen on summation were examined using small current pulses (250 pA, 20 ms) injected into B8 to simulate synaptic currents without firing the presynaptic neuron B20 (Fig. 10). It was previously shown that trains of such pulses can exhibit "summation" when applied within the physiological firing frequency of B20 (Fig. 5). Application of baclofen (1 mM) decreased the rate of decay of voltage deflections such that successive pulses within a train were incrementally prolonged and their summed depolarization of B8 was increased (Fig. 10B–D). As the effect of baclofen developed, the cumulative depolarization of pulse trains produced firing of B8 at progressively earlier phases (Fig. 10C, D).
Figure 10. Baclofen increases the decay time of voltage deflections produced by current pulses in B8. (A) A train of current pulses (5 ms, 0.25 nA) was injected into B8 to mimic the EPSPs that are produced by a burst of B20 impulses. (B–D) In the presence of baclofen (1 mM), the decay times of the voltage deflections became successively prolonged. Pulse trains that failed to reach the threshold of B8 under control conditions became capable of producing action potentials at progressively earlier phases of their duration.
Together, these results demonstrate that GABA, acting via GABAB-like receptors, enhances facilitation and summation from B20 to B8 but not to B16. The increase in B20-to-B8 facilitation appears to reflect presynaptic properties that are independent of the postsynaptic membrane potential. The increase in summation can be attributed to postsynaptic actions in B8 that produce decreased rates of decay in successive EPSPs.
Discussion
In this study, signaling from the influential buccal interneuron B20 was progressively biased toward one of its followers during trains of action potentials. Our observations indicate that this bias resulted from presynaptic and postsynaptic mechanisms acting in a concerted fashion (Fig. 11A). Both contributions to this bias were amplified by modulatory effects of GABA, which increased facilitation and summation in a target-specific fashion (11B). These findings predict that, excluding additional factors, patterned firing of B20 will be more effective in producing excitation of B8 than of B16. This was in fact observed with simultaneous recording of the two motor neurons when trains of impulses were produced in B20 (Fig. 11C).
Figure 11. Divergent short-term synaptic plasticity and modulatory metaplasticity in the rapid excitatory signaling from B20 to B16 and B8. (A) Schematic of divergent excitatory signaling from B20 to the two radula closer motor neurons B16 and B8. Short-term facilitation and summation were greater in the rapid EPSPs to B8 (labeled with larger type). Differences in facilitation are attributed to target-specific properties of the B20 synapses. Differences in summation are attributed to dissimilar membrane properties of the two postsynaptic motor neurons. (B) Proposed modulatory effects of GABA on the divergent signaling of B20. Actions of GABA were not detected on either facilitation or summation of the EPSPs from B20 to B16. In B8, both of these short-term increases in the synaptic efficacy of signaling of from B20 were enhanced by GABA and baclofen. GABAB effects on facilitation are proposed to reflect actions on the B20-to-B8 presynaptic terminals. Effects on summation are proposed to reflect GABAB-like actions on the membrane properties of B8. (C) When repetitive trains of B20 impulses were produced by injection of current pulses (bottom record), facilitation and summation of EPSPs contributed to firing B8 while responses in B16 remained subthreshold. Recording of the impulse in B8 was truncated by the data acquisition system.
Previous investigations localized markers for catecholamines and GABA to B20 (Díaz-Ríos et al., 2002) and showed that its fast EPSPs to both B16 and B8 were mediated by dopamine (Díaz-Ríos and Miller, 2005). While it is intriguing to consider that GABA could be acting as a cotransmitter at these synapses, its release from B20 remains to be demonstrated, and its source is therefore not specified in our present schema (Fig. 11B). However, whether the modulation of signaling from B20 is homosynaptic or heterosynaptic in origin, its presence in an interneuron that is embedded within the buccal CPG would classify it as an intrinsic form of modulation (Katz et al., 1994; Katz and Frost, 1996; see Introduction). As such, its magnitude can be expected to both reflect and influence the degree to which the overall system is activated.
A type of modulation with formal similarities to that observed in this study has been demonstrated in the circuit mediating the siphon withdrawal reflex of Aplysia (Fischer et al., 1997). In that system, serotonin acting in a heterosynaptic fashion attenuated the ability of an identified inhibitory neuron (L30) to display short-term synaptic enhancement. This action was designated "modulatory metaplasticity" to distinguish it from previously described homosynaptic forms of metaplasticity in which a neuron’s firing history influenced its ability to express plasticity (Abraham and Bear, 1996). If GABA is released when B20 fires, and if that synaptically released GABA is capable of influencing subsequent rapid dopaminergic signaling, then both of these terms would be applicable to B20-to-B8 signaling during impulse trains. In the context of the existing nomenclature, such regulation would be designated "homosynaptic modulatory metaplasticity" (Fig. 11B).
Divergent synaptic plasticity
Branch, or target-specific, synaptic plasticity, such as that observed in the signaling from B20 to its two follower motor neurons, appears to be used extensively to achieve divergent channeling of information flow in motor systems. In the neuromuscular systems of crustaceans, where a small number of neurons control multiple muscles, terminals of an individual motor neuron can exhibit wide ranges of facilitation or depression (Atwood, 1967; Bittner, 1968). In an extreme case, the terminals of a single stomatogastric motor neuron in the lobster were shown to exhibit facilitation in one muscle and depression in another muscle (Katz et al., 1993).
Within the Aplysia buccal ganglion, diverse manifestations of differential synaptic plasticity can contribute to CPG function. Gardner and Kandel (1977) identified a diphasic synaptic potential (EPSP followed by an IPSP) originating from the cholinergic interneuron B4/5. Repeated firing of B4/5 preferentially reduced the magnitude of the hyperpolarizing component of this PSP, eventually resulting in a monophasic depolarization. In that instance, differential rates of synaptic depression were attributed to properties of distinct postsynaptic acetylcholine receptors that exhibited different rates of desensitization (Gardner and Kandel, 1977). Another form of differential synaptic plasticity was recently shown to occur in the signaling of buccal interneuron B65, where EPSPs exhibit depression in one follower neuron (B4/5) and facilitation in a second (B8; Kabotyanski et al., 1998). Interestingly, B65 shares the GABA-DA neurotransmitter phenotype with B20, the subject of this investigation (Díaz-Ríos et al., 2002; Due et al., 2004).
Complementary pre- and postsynaptic modulation of plasticity
Previously, it was found that GABA, acting via GABAB-like receptors, produced a small depolarization and increase in the input resistance and excitability of B8 (Díaz-Ríos and Miller, 2005). GABA and baclofen were also found to increase the amplitude of EPSPs evoked in B8 by single impulses in B20. The present investigation demonstrated that GABA can also enhance signaling from B20 to B8 by increasing facilitation and summation. Additional mechanisms, including postsynaptic interactions between the neurotransmitters present in B20, may also be operating to modify these signals. Recently, it was shown that GABA and baclofen increase inward currents produced by dopamine in B8 (Svensson et al., 2004). If GABA is in fact co-released with dopamine from B20, then such synergistic influences could contribute to the enhancement of the B20-to-B8 EPSP that is observed during impulse trains.
Presynaptic modulation.
GABAB receptors have been shown to regulate transmitter release in a range of systems and species (see review by Bettler et al., 2004), including several invertebrates (Miwa et al., 1990; Parnas et al., 1999; Gutovitz et al., 2001). Prior to the recognition of GABA receptor subtypes, Phillipe and coworkers (1981) found that baclofen (3–5 x 10–5 M) altered synaptic plasticities in the Aplysia abdominal ganglion. In that study, the effects of baclofen were thought to be indirect and were attributed to its ability to liberate biogenic amines (Philippe et al., 1981). In the system studied in the present investigation, the concentrations of GABA and baclofen required to produce detectable effects were considerably higher than are normally found to activate GABAB receptors. However, if GABA originates from the terminals of B20 itself, it is likely to be capable of achieving high concentrations at or near the sites of dopamine release.
We propose that the GABA-induced increases in facilitation reflect presynaptic actions that modify the history-dependence of neurotransmitter release from B20. Similar presynaptic modulatory actions have been demonstrated with aminergic neuromodulators in Aplysia central synapses (Tremblay et al., 1976; Woodson et al., 1976; Newlin et al., 1980). Their possible behavioral significance is best understood in the circuits that mediate defensive reflexes, where presynaptic modulation is implemented by serotonin acting in a heterosynaptic fashion (Carew and Kandel, 1974; Kandel and Schwartz, 1982; Byrne and Kandel, 1996).
Postsynaptic modulation.
Our findings indicate that GABA, acting via GABAB-like receptors on B8, enhances summation of the B20-to-B8 EPSP. This modulatory action did not occur when the membrane potential of B8 was set to hyperpolarized levels. GABA did, however, enhance the "summation" of voltage deflections produced by current pulses injected into B8 at its resting Vm. Its effects appear, therefore, to reflect an action on membrane properties of B8 that are operative near its resting membrane potential. We propose that the progressive advance of the B8 Vm into a region of increased resistance during a train of EPSPs contributes to the enhanced summation of successive EPSPs and that the modulatory effects of GABA reflect an action that augments this intrinsic property.
Several buccal interneurons and sensory neurons converge upon B8 and B16, and their synaptic actions on these motor neurons have been related to their ability to specify motor programs (Kabotyanski et al., 1998; Nargeot et al., 1999b; Jing and Weiss, 2001; Morgan et al., 2002; Jing et al., 2003; Proekt et al., 2004). A previous investigation demonstrated a nonlinearity in the membrane properties of B8 that could modify EPSPs originating from the identified mechanosensory neuron B21 (Klein et al., 2000). In that study, the amplitude and time constant of decay of the B21-to-B8 EPSP were both decreased when the membrane potential of B8 was set to hyperpolarized levels. The nonlinear properties of B8 were likened to the "anomalous rectification" that occurs in the serotonergic metacerebral cell (MCC) of Aplysia (Kandel and Tauc, 1966). In the MCC, anomalous rectification strongly influenced the ability of repetitive EPSPs to summate (Kandel and Tauc, 1966). In agreement with observations made in the MCC, we found that the effect of the B8 membrane potential on the rate of decay of the B20-to-B8 EPSP was substantially greater than its effect on the magnitude of this EPSP.
Divergent modulation of plasticity—potential contributions to behavioral plasticity
The phase of radula closure with respect to its protraction and retraction is the operational parameter that distinguishes ingestive from egestive feeding behaviors (Kupfermann, 1974; Morton and Chiel, 1993a, Morton and Chiel, 1993b; Cropper et al., 2004). In experiments with both in vivo and in vitro preparations, radula closure is commonly monitored by observing the phase and pattern of B8 firing (Church and Lloyd, 1994; Nargeot et al., 1997, Nargeot et al., 1999a, Nargeot et al., 1999b; Kabotyanski et al., 1998; Sweedler et al., 2002). The influence of B20 on the buccal CPG is best understood in the context of egestive motor patterns produced by stimulation of cerebral-buccal command neurons (Jing and Weiss, 2001) or the esophageal nerve (Proekt et al., 2004). In both instances, the direct B20-to-B8 EPSP is thought to specify egestive motor programs by promoting radula closure during protraction. The degree to which the forms of synaptic enhancement examined in this study contribute to such motor-pattern specification remains uncertain. Several observations suggest that additional more persistent forms of plasticity also modify the signaling of B20 to B8 (Teyke et al., 1993; Jing and Weiss, 2001; Proekt et al., 2004; our observations).
The initiation of a feeding bout in Aplysia is characterized by a progressive increase in the frequency and intensity of biting, a feature that is thought to reflect food-induced arousal (Kupfermannn et al., 1979; Rosen et al., 1989). Transitions of buccal motor programs also occur in a graded fashion and exhibit some inertia (Kabotyanski et al., 1998; Brezina et al., 2003a, Brezina et al., 2003b; Proekt et al., 2004). A progressive increase in the efficacy of excitatory signaling from B20 to B8 was recently shown to accomplish the graded transition of the buccal CPG toward egestive motor patterns (Proekt et al., 2004). Motor-pattern warm-up and transitions are likely to require that radula closer motor neurons be recruited in a specific sequence (Brezina et al., 2003a, Brezina et al., 2003b; Zhurov et al., 2005; Ye et al., 2006). The divergent short-term synaptic plasticities from B20 to B16 and B8, and their differential modulation by GABA, could contribute to the effective and adaptive onset and transitioning of repetitive motor patterns.
Recent investigations have increased our appreciation for the scope and complexity of neural mechanisms that contribute to relatively simple forms of behavioral plasticity in Aplysia (Glanzman, 1995; Byrne and Kandel, 1996; Martin et al., 2000). The mechanisms underlying learning and memory in the polymorphic circuits that produce repetitive behaviors are likely to exhibit comparable or even greater levels of distribution and complexity (see Benjamin et al., 2000). Correlates of associative conditioning in molluscan feeding circuits have been shown to occur in the sensory pathways that mediate food detection (Morielli et al., 1986; Staras et al., 1999; Lechner et al., 2000a; Mozzachiodi et al., 2003), in the signaling from command-like interneurons to pattern-initiating CPG elements (Kovac et al., 1986; Lechner et al., 2000b; Straub et al., 2004), and in the biophysical properties of key buccal interneurons (Nargeot et al., 1999a, Nargeot et al., 1999b; Brembs et al., 2002). The contribution of plastic synaptic signaling to the motor neurons themselves, a major determinant of non-CPG-mediated behavioral plasticity (see contributions to this symposium), has received less scrutiny. This report aims to stimulate further investigation into modifications of signaling to the final common pathway that executes such CPG-mediated motor programs when the experience of an animal causes it to adjust its actions.
Acknowledgments
Supported by the National Science Foundation: CAREER Award: IBN-9722349; National Institutes of Health DRS RCMI RR-03051, NIGMS MBRS: GM-08224, and NIMH MRISP MH48190; and funds from the University of Puerto Rico Medical Sciences Campus Deanship of Biomedical Sciences.
Footnotes
Received 7 November 2005; accepted 23 January 2006.
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