Different Roles of Neurons B63 and B34 That Are Active During the Protraction Phase of Buccal Motor Programs in Aplysia californica

Itay Hurwitz1, 2, Irving Kupfermann2, and Abraham J. Susswein1

1 Department of Life Sciences, Bar-Ilan University, Ramat-Gan 52 900, Israel; and 2 Center for Neurobiology and Behavior, College of Physicians and Surgeons, Columbia University, New York, New York 10032

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Hurwitz, Itay, Irving Kupfermann, and Abraham J. Susswein. Different roles of neurons B63 and B34 that are active during the protraction phase of buccal motor programs in Aplysia californica. J. Neurophysiol. 78: 1305-1319, 1997. The buccal ganglion of Aplysia contains a central pattern generator (CPG) that organizes sequences of radula protraction and retraction during food ingestion and egestion. Neurons B63 and B34 have access to, or are elements of, the CPG. Both neurons are depolarized along with B31/B32 during the protraction phase of buccal motor programs. Both cells excite the contralateral B31/B32 neurons and inhibit B64 and other neurons active during the retraction phase. B63 and B34 also both have an axon exiting the buccal ganglia via the contralateral cerebrobuccal connective. Despite their similarities, B63 and B34 differ in a number of properties, which reflects their different functions. B63 fires during both ingestion and egestion-like buccal motor programs, whereas B34 fires only during egestion-like programs. The bilateral B63 neurons, along with the bilateral B31 and B32 neurons, act as a single functional unit. Sufficient depolarization of any of these neurons activates them all and initiates a buccal motor program. B63 is electrically coupled to both the ipsilateral and the contralateral B31/B32 neurons but monosynaptically excites the contralateral neurons with a mixed electrical and chemical excitatory postsynaptic potential (EPSP). Positive feedback caused by electrical and chemical EPSPs between B63 and B31/B32 contributes to the sustained depolarization in B31/B32 and the firing of B63 during the protraction phase of a buccal motor program. B34 is excited during the protraction phase of all buccal motor programs, but, unlike B63, it does not always reach firing threshold. The neuron fires in response to current injection only after it is depolarized for 1-2 s or after preceding buccal motor programs in which it is depolarized. Firing of B34 produces facilitating EPSPs in the contralateral B31/B32 and B63 neurons and can initiate a buccal motor program. Firing in B34 is strongly correlated with firing in the B61/B62 motor neurons, which innervate the muscle (I2) responsible for much of protraction. B34 monosynaptically excites these motor neurons. B34 firing is also correlated with firing in motor neuron B8 during the protraction phase of a buccal motor program. B8 innervates the I4 radula closer muscle, which in egestion movements is active during protraction and in ingestion movements is active during retraction. B34 has a mixed, but predominantly excitatory, effect on B8 via a slow conductance-decrease EPSP. Thus firing in B34 leads to amplification of radula protraction that is coupled with radula closing, a pattern characteristic of egestion.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Repetitive movements in many animals are organized by central pattern generators (CPGs), circuits organizing the pattern and timing of an activity (Getting 1989; Pearson 1993; Selverston and Moulins 1985). The buccal ganglion of Aplysia contain a CPG controlling feeding. This CPG is of particular interest, because Aplysia feeding is a model system for examining how a complex behavior is regulated by motivation and learning (Chiel and Susswein 1993; Kupfermann et al. 1991; Susswein et al. 1986). Identifying neurons that are part of the CPG can provide insight into how these neurons may be modified by these processes. Control of feeding in other gastropod mollusks is also being studied (Arshavsky et al. 1989a,b; Cappell et al. 1989; Croll et al. 1985; Delaney and Gelperin 1990; Elliott and Benjamin 1985; Kovac et al. 1986; Quinlan and Murphy 1991; Quinlan et al. 1995; Yeoman et al. 1995), allowing comparison with the regulation of feeding in Aplysia.

Aplysia feeding is composed of appetitive movements, by which animals locate food, and three subsequent consummatory movements: 1) biting, by which food enters the mouth; 2) swallowing, which transports food into the gut; and 3) rejection, which pushes food out of the mouth (Hurwitz and Susswein 1992; Hurwitz et al. 1996; Weiss et al. 1986a). These movements consist of a protraction and retraction of the toothed radula, coupled with opening and closing of the radula halves. In ingestion (biting and swallowing), the food moves inward. The radula halves open during a relatively weak protraction and close on the food during a powerful retraction. In rejection, protraction causes outward food movement. The radula halves close on food during a powerful protraction, and a weak retraction with the radula halves open returns the radula to rest (Morton and Chiel 1993a,b). Switching between ingestion and egestion is accomplished by changing the relative amplitude of the protraction and retraction phases and by changing the coupling between a protraction-retraction sequence and the accompanying radula opening and closing (Church and Lloyd 1994; Morton and Chiel 1993a). Consummatory movements are correlated with buccal motor programs that can be recorded in intact animals as well as in reduced preparations. During a complete buccal motor program, activity corresponding to radula protraction and retraction can be monitored from many neurons as well as by recording from peripheral nerves (Hurwitz et al. 1996; Morton and Chiel 1993a; Susswein et al. 1996). The activity of protractor, retractor, and closer motor neurons can be used to distinguish between different types of buccal motor programs.

A number of neurons that are components of the buccal ganglia CPG have been identified (Baxter and Byrne 1991; Hurwitz and Susswein 1996; Kabotyansky et al. 1994; Plummer and Kirk 1990; Susswein and Byrne 1988; Teyke et al. 1993). Of particular interest are neurons B31/B32, which are active during the protraction phase of feeding (Hurwitz et al. 1996) and can induce repetitive buccal motor programs (Susswein and Byrne 1988). Activity in the B31/B32 somata is characterized by a sustained depolarization of 30-40 mV (Susswein and Byrne 1988), with various fast potentials superimposed on the depolarization. Some of the fast potentials represent spikes in the B31/B32 axon that fail to invade the soma (Hurwitz et al. 1994); others represent spikes in strongly coupled neurons, such as B33 (Susswein and Byrne 1988); still others may represent excitatory postsynaptic potentials (EPSPs) from neurons that are presynaptic to B31/B32. Understanding how B31 and B32 function requires one to identify additional neurons that activate them and that are active with them. In this paper we explore the properties of two such neurons, B34 and B63. Although both neurons have similar morphological features, they differ greatly in their properties and firing patterns and presumably in their functions. B63 always fires along with the sustained depolarization in B31/B32. Activity in B31/B32 occurs during the protraction phase of biting, swallowing, and rejection movements (Hurwitz et al. 1996), suggesting that B63 is also active during all of these movements. However, B34 fires during the protraction phase of only a subset of buccal motor programs. We present evidence that B34 may function in producing an egestion type of motor program.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Animals

In Ramat Gan, experiments were performed on Aplysia californica weighing 50-100 g that were purchased from T. Capo, University of Miami (Miami, FL). Animals were maintained in 940-l aquaria of filtered Mediterranean seawater kept at 17°C. In New York, experiments were performed on 100- to 250-g A. californica (Marinus, Long Beach, CA) maintained in 600-l tanks of artificial seawater at 14°C.

Physiological media and procedures

Animals were immobilized by injection of 25-50% of the body volume of isotonic MgCl2 into the hemocoel. The buccal ganglia (attached to the cerebral ganglion or to the I2 muscle) (see Hurwitz et al. 1994) were then removed and placed in a chamber containing 70% filtered seawater and 30% isotonic MgCl2. The connective tissue sheath over the caudal surface of the buccal ganglia was then surgically removed. After desheathing, the bathing solution was replaced with artificial seawater (composition, in mM: 450 NaCl, 10 KCl, 22 MgCl2, 33 MgSO4, 10 CaCl2, 10 glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, working pH 7.4). Intracellular recording and stimulation were performed at room temperature (22-24°C) with the use of 2- to 10-MOmega electrodes filled with 3 M potassium acetate. In some experiments the bathing medium was changed. The altered media used were composed of (in mM): 3 × Ca2+, 3 × Mg2+---270 NaCl, 6 KCl, 120 MgCl2, 33 MgSO4, and 30 CaCl2; Ca2+ free---450 NaCl, 10 KCl, 22 MgCl2, 33 MgSO4, and 0 CaCl2; 0.1 × Ca2+, 4 × Mg2+---250 NaCl, 6 KCl, 175 MgCl2, 33 MgSO4, and 1 CaCl2; Ca2+ free, 10 mM Co2+---450 NaCl, 10 KCl, 22 MgCl2, 33 MgSO4, 0 CaCl2, and 10 CoCl2.

In some experiments, cells were filled with cobalt hexamine chloride (Kodak) prepared as a saturated solution. The dye was injected iontophoretically with the use of 500-ms depolarizing pulses at 1 Hz, with currents ranging from 1 to 20 nA. Preparations were developed with hydrogen sulfide, fixed in 4% glutaraldehyde, dehydrated and cleared as whole mounts, and then drawn with the use of an Olympus compound microscope equipped with camera lucida.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Intracellular stimulation of the B31/B32 neurons elicits a patterned burst of activity in the buccal ganglia, probably because these neurons are part of a CPG (Susswein and Byrne 1988). Neurons exciting B31/B32 may also be part of the CPG or may have a commandlike function. We have characterized two such neurons, B63 and B34. B63 had not been previously identified. B34 had been previously described (Susswein and Byrne 1988), but its ability to initiate patterned bursts had not been found. B63 was recorded in 22 preparations, whereas B34 was recorded in >45 preparations.

Morphology of B63 and of B34

Similar to most neurons in the buccal ganglia, both B63 and B34 are bilaterally symmetrical, with one such neuron in each hemiganglion. To visualize the morphology of B63 and B34, their somata were filled with cobalt hexamine chloride (B63, n = 5; B34, n = 13). The somata of B63 and B34 are located laterally in the ganglia (Fig. 1). B63 is a small neuron located below the caudal surface of the ganglion, about halfway between B1 and B8. B34 is located just adjacent to B31/B32, usually somewhat medial to these cells. B63 and B34 both have axons that travel medially and cross the ganglion, entering and crossing the buccal commisure to the contralateral buccal ganglion and exiting the contralateral ganglion via the cerebrobuccal connective. Synaptic connections of both B63 and B34 to neurons in the cerebral ganglion have been found, and these will be described elsewhere (Hurwitz and Kupfermann, unpublished data). Thus both B63 and B34 are buccal-cerebral interneurons. However, they differ from buccal-cerebral interneurons that have been described previously (Chiel et al. 1988; Rosen et al. 1991b; Teyke et al. 1993) in having exclusively contralateral axons. B63 neurites are present in the region of the ipsilateral and contralateral B31/B32 neurons, as well as in the region of the ipsilateral and contralateral B4/B5 neurons, which are adjacent to the previously identified (Teyke et al. 1993) pattern-initiating, dopaminergic B20 neurons (Fig. 1B). The B34 axon gives off numerous neurites in both the ipsilateral and contralateral hemiganglion, as well as in the buccal commissure (Fig. 1C). The axon also gives off many more neurites in the contralateral hemiganglion than B63 does before it enters the cerebrobuccal connective (Fig. 1C).


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FIG. 1. Morphology of B63 and B34. Drawings are from caudal surface. A: map of buccal ganglia showing locations of B63, B34, and other neurons discussed in this paper. B and C: morphologies of B63 and B34, respectively. Both neurons have an axon that crosses to contralateral buccal hemiganglion and then leaves buccal ganglia via contralateral cerebrobuccal connective. For B63, note that there are neurites adjacent to ipsilateral and contralateral B31/B32, to B20 just medial to B4, and to B4/B5 areas (&cjs0681;). For B34, note presence of neurites adjacent to B34 soma, as well as in ipsilateral and contralateral ganglion, and within buccal commissure. BN-1, BN-2, BN-3: buccal nerves 1-3, according to terminology of Gardner (1971). ESO, esophageal nerve; CBC, cerebrobuccal connective; RN, radula nerve. S1 and S2, groups of small cells (Fiore and Meunier 1979).

B63 excites CPG elements and drives buccal motor programs

In intact, behaving animals, B31/B32 activity is correlated with protraction of the radula, which is followed by radula retraction, during which B31/B32 is silent (Hurwitz et al. 1996). In isolated buccal ganglia preparations, buccal motor programs consisting of cycles of protraction-retraction sequences can be monitored by recording from many neurons (Church and Lloyd 1994). During such programs, B31/B32 are depolarized and active during the protraction phase (Fig. 2A), whereas other neurons, such as B64 and B4, fire during the retraction phase. B31/B32 shows a large inhibitory postsynaptic potential (IPSP) that is partially caused by B64 during the retraction phase (Hurwitz and Susswein 1996). The sustained depolarization and the subsequent hyperpolarization of B31/B32 can be used as monitors of the protraction and retraction phases of a program.


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FIG. 2. A: spontaneous buccal motor programs recorded in buccal ganglia are divided into protraction and retraction phases. Depolarization in B31/B32 corresponds to protraction phase, whereas retraction is characterized by hyperpolarization in B31/B32 and firing in B64 and in B4 (Hurwitz et al. 1996). B: simultaneous recording from 3 B31/B32 neurons (2 from right buccal ganglion, 1 from left) at start of spontaneous buccal motor program. Note that program begins with barrage of excitatory postsynaptic potentials (EPSPs) that are synchronous in 2 neurons recorded in right hemiganglion, whereas similar but asynchronous EPSP is seen in neuron recorded from left hemiganglion. Also note that blocked axon spikes (right-arrow) begin to be seen only after early EPSPs. EPSPs are attributed to activity of B63.

A barrage of fast depolarizing potentials is prominently seen at the start of the B31/B32 depolarization signaling the protraction phase. As this depolarization proceeds, the potentials increase in frequency. Somewhat after these potentials, larger (~10 mV), fast depolarizations appear, which represent blocked axon spikes in B31/B32 (Hurwitz et al. 1994). Simultaneous recordings from two B31/B32 neurons in the same hemiganglion and from one B31/B32 neuron in the opposite hemiganglion (Fig. 2B) showed that the initial depolarizing potentials are synchronous in the same hemiganglion, whereas similar but nonsynchronous potentials are recorded in the opposite hemiganglion. These depolarizing potentials represent EPSPs from B63. A single B63 neuron is responsible for driving both B31 and B32 in each hemiganglion.

CONNECTIONS FROM B63 TO B31/B32. B63 and B31/B32 are active synchronously during buccal motor programs (Fig. 3A). However, B63 differs from B31/B32 in that it displays conventional action potentials. During the retraction phase of a program B63 shows a hyperpolarization synchronous with that in B31/B32.


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FIG. 3. Relationship between B63 and B31/B32. A: synchronous activity recorded during spontaneous buccal motor program in right and left B31/B32 neuron and in left B63 neuron. EPSPs in right B31/B32 are correlated with action potentials in left B63. Activity in left B31/B32 neuron begins somewhat later, and EPSPs early in activity of this neuron are not related to spikes in B63 neuron. B: depolarization of B63 (up-arrow  down-arrow ) initiates buccal motor programs that can be monitored in both B63 and B31/B32. C: brief depolarization of B63 elicits 1-for-1 EPSPs in B31/B32.

B63 spikes were typically synchronous with fast potentials in the contralateral B31/B32 neurons (Fig. 3A). In 20 of 22 preparations B63 showed exclusively contralateral synchrony; in a single preparation the potentials were bilateral, and in a second preparation the synchrony was ipsilateral. In the example shown in Fig. 3A, a spontaneous buccal motor program was recorded from a right and a left B31/B32 neuron and from the right B63. Spikes in the B63 neuron are followed by one-for-one EPSPs in the contralateral B31/B32 neuron, whereas the ipsilateral B31/B32 shows EPSPs of a similar waveform that are not synchronized with B63 spikes. Presumably, these were correlated with firing of the contralateral B63 neuron, which was not impaled.

Sustained stimulation of B63 was able to also elicit repetitive bursts of activity (Fig. 3B). Brief stimulation of B63 elicits a few spikes, which elicit one-for-one EPSPs in the contralateral B31/B32 (Fig. 3C).

Many neurons fire during buccal motor programs, and their firing can be used to monitor a program. However, not all neurons are equally reliable monitors. Some neurons fire during all programs, whereas others fire only during a fraction of them. Firing in B31/B32 and B64 is a highly reliable monitor of buccal motor programs, whereas firing in B4/B5, B61/B62, and B34 is variable (Hurwitz and Susswein 1996; Hurwitz et al. 1996), although all of these neurons display slow potentials in phase with a program. In the absence of experimenter intervention, firing in the B63 neurons is as reliable a monitor of a buccal motor program as is activity in B31/B32 and B64.

B63 AND B31/B32 ARE ELECTRICALLY COUPLED. Depolarizing either B31/B32 or B63 elicits buccal motor programs in which both B31/B32 and B63 are active, suggesting that B31/B32 and B63 are mutually excitatory. We examined the excitation of B31/B32 to B63.

Brief depolarization of a single B31/B32 neuron in a manner similar to that initiating a buccal motor program causes depolarization of both the ipsilateral and the contralateral B63 neuron (Fig. 4A). Depolarization is seen before the start of spiking in the B31/B32 axon, indicating that conventional direct chemical EPSPs from B31/B32 to B63 cannot explain the excitation. Note that in the example shown, the depolarization of B31/B32 produced many small fast potentials that were unrelated to activity in B63. We hypothesized that these potentials may be attributed to the firing of identified neuron B33, which is strongly electrically coupled to B31/B32 (Susswein and Byrne 1988) and which has a lower threshold for firing in response to depolarization of the B31/B32 soma than does the B31/B32 axon (see Fig. 11 in Hurwitz et al. 1994). Because recordings from B33 and B63 showed that spikes in B33 did not cause EPSPs in B63 (not shown), the coupling from B31/B32 to B33 could not account for the excitation of B63. Also, note that in this figure the spikes elicited in the contralateral B63 neuron did not elicit noticeable EPSPs in the B31/B32. The EPSPs were presumably masked by the strong depolarization of B31/B32. Injecting depolarizing or hyperpolarizing currents into a B31/B32 neuron, or into a B63 neuron, shows that the B31/B32 neurons are electrically coupled to both the right and left B63 neurons (Fig. 4B). The coupling ratio between the ipsilateral B31/B32 and B63 neurons is ~5:1, whereas the coupling between contralateral B31/B32 and B63 neurons is ~10:1. In addition, coupling between the right and left B63 cells is ~30:1. Previous data have shown that the ipsilateral B31 and B32 neurons are coupled with a ratio of 2:1 (Susswein and Byrne 1988).


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FIG. 4. Coupling between B31/B32 and B63. A: depolarization of B31/B32 (up-arrow  down-arrow ) leads to depolarization and firing of both ipsilateral and contralateral B63 neurons. B: depolarizing and hyperpolarizing current pulses in single B31/B32 or B63 neuron cause bilateral effects. B1: depolarization of B31/B32 neuron (up-arrow  down-arrow ) causes bilateral depolarization of B63 neurons. B2: hyperpolarization of B63 neuron causes bilateral hyperpolarization of B31/B32 neurons.


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FIG. 11. Amplification of excitation onto B31/B32 is via summation and facilitation. A: B34 neuron was stimulated with 6 identical depolarizing current pulses (up-arrow  down-arrow  underneath B34 trace). These elicited progressively more action potentials, which in turn produced progressively larger depolarization of B31/B32. Increased depolarization reflected temporal summation as well as facilitation of EPSPs. B: in 12 runs performed in 4 preparations, we measured the number of spikes elicited by each of 5 current injections (15 nA for 0.5 s every 1 s) and amplitude of EPSPs produced in B31/B32 neuron by each spike. For each run, EPSP amplitudes were normalized to amplitude of 1st EPSP elicited by 1st spike-eliciting stimulus. All runs in a single preparation were then averaged, and mean ± SE of EPSP amplitude for all 4 preparations was calculated. For each successive EPSP, mean normalized amplitude of EPSP is graphed, along with mean ± SE (vertical bars). Also shown is number of runs (N) in which each successive EPSP was seen. For example, during 5th stimulus 1st 5 EPSPs were seen during all 12 runs. Sixth EPSP was seen during 8 runs, 7th EPSP during 6 runs, 8th EPSP during 4 runs, and 9th EPSP during 2 runs. Data show progressive increase in number of spikes elicited and in amplitude of EPSPs produced in B31/B32 by these spikes. Note that decreases in EPSP amplitude seen toward end of stimuli 4 and 5 arise because preparations showing relatively less facilitation responded to stimulus with more spikes than did preparations displaying more prominent facilitation. Thus only relatively weakly facilitating preparations are represented by higher EPSP numbers.

It was difficult to record simultaneously from the right and left B63 neurons, because they are relatively small. Figure 4A is one of the few examples in which such a recording was obtained. In this record the first action potential in the left B63 neuron caused an EPSP in the right B63 neuron. Subsequent action potentials caused EPSPs and firing, indicating that the B63 neurons are mutually excitatory. We did not explore whether this excitation has a chemical component in addition to the electrical coupling.

CONTRALATERAL EPSPS HAVE BOTH CHEMICAL AND ELECTRICAL COMPONENTS. Coupling is somewhat stronger between the ipsilateral B63 and B31/B32 neurons than between the contralateral neurons. Nonetheless, B63 elicits one-for-one EPSPs only in the contralateral B31/B32. This could be explained in two ways. 1) The fast contralateral EPSP is electrical. However, the site of coupling is distant from the B63 soma that is impaled, probably in the contralateral buccal ganglion. Coupling in the contralateral ganglion may be sufficient to account for the fast EPSP, but cannot be accurately measured by recording from the B63 and B31 somata located in opposite hemiganglia (see Fig. 19B). 2) The connection from B63 to the contralateral B31/B32 neurons represents a chemical EPSP.


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FIG. 19. A: diagram summarizing synaptic connections between neurons discussed in this paper. Note that weak coupling between B34 and B31/B32 (Susswein and Byrne 1988) is not shown. Also, amplitude of connection is generally not indicated. One exception is that mixed excitatory-inhibitory synapse from B34 to B8 is shown with small inhibition and large excitation to emphasize that connection is primarily excitatory. In addition, there is no indication of whether connection is ipsilateral, contralateral, or bilateral. B: diagram showing relationships between bilateral B63 and B31/B32 neurons. Open triangles: excitation. Filled circles: inhibition. Electrical connections are shown by resistors.

Sustained depolarization of B63 that caused a series of action potentials revealed a weak facilitation (~30% of the original size) in the EPSPs recorded in a contralateral B31/B32, suggesting that the EPSP is chemical (Fig. 5A). Additional data in support of a chemical EPSP came from experiments in which the preparation was bathed in 3 × Ca2+, 3 × Mg2+ seawater (not shown). In this solution, EPSPs were approximately twice the size of controls, presumably because of increased facilitation caused by the increase in Ca2+ concentration. However, when the buccal ganglia were bathed in Ca2+-free, 10 mM Co2+ seawater (Co2+ replaced the extracellular Ca2+), thereby blocking chemical synaptic transmission (synapses from B63 to other neurons in the buccal ganglia were blocked), one-for-one EPSPs were still seen (Fig. 5B), suggesting that at least part of the EPSP is also electrical. The nature of the EPSP was more easily seen when the buccal ganglia were bathed in a Ca2+-free solution in which the Ca2+ was not replaced by another cation. The residual Ca2+ in the preparation is sufficient to allow some chemical synaptic transmission, although much of it is blocked. Comparing the first EPSP elicited by B63 with subsequent EPSPs showed that the first EPSP had only a single component, whereas later EPSPs seemed to be composed of two components. The earlier component is presumably electrical, whereas the later component is a facilitated chemical EPSP (Fig. 5C). Thus the connection from B63 to seems to have both electrical and chemical components, with the chemical component showing a modest facilitation in response to repeated spikes.


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FIG. 5. EPSP from B63 to contralateral B31/B32 is partially chemical and partially electrical. A: EPSPs are elicited in B31/B32 by spikes in contralateral B63 in normal saline (ASW, artificial seawater). B: EPSPs were also seen in solution in which Ca+2 was replaced by Co+2, thereby blocking chemical synaptic transmission. C: in Ca+2-free solutions a series of EPSPs was elicited. First and last EPSPs from series were superimposed, showing that EPSP has 2 components: an early component that is presumably electrical and a later component that shows moderate facilitation and is likely to be a chemical EPSP.

POSITIVE FEEDBACK AND TEMPORAL SUMMATION CONTRIBUTE TO THE SUSTAINED DEPOLARIZATION OF B31/B32. The reciprocal excitation caused by electrical and chemical synaptic connections between the bilateral B63 and B31/B32 neurons suggests a possible explanation for the ability of a single B63 or B31/B32 neuron to activate the entire bilateral B63-B31/B32 complex and for the patterns of activity observed in this complex during the protraction phase of buccal motor programs. Activating a single B63 neuron (either by direct depolarization or via a coupled B31/B32 neuron) causes it to fire, thereby causing EPSPs in the contralateral B31 and B32 neurons. The relatively high frequency of firing in B63 leads to summation of these EPSPs, which gives rise to the 30-mV sustained depolarization seen in B31/B32 when it is active. This sustained depolarization causes depolarization of the electrically coupled contralateral B63 neuron and causes it to fire, and this firing in turn produces a gradually increasing depolarization in the ipsilateral B31/B32 neurons. Thus all six neurons in the B31/B32-B63 complex become active together.

A number of observations suggested that positive feedback provided by coupling and temporal summation of the EPSPs from B63 to B31/B32 are not the only processes contributing to the activity pattern observed in B63 and in B31/B32 during their sustained activity. High-gain recordings from B31/B32 before and during the start of a buccal motor program showed that at rest B31/B32 receives a steady barrage of small (250-500 µV) IPSPs. Cessation of these IPSPs is often the first sign that a buccal motor program has been initiated, indicating that disinhibition of B31/B32 can contribute to the initiation of the sustained depolarization in B31/B32. In addition, depolarization of B63 or of B31/B32 initiates a slow depolarization in some of the cells in the B31/B32-B63 complex or in cells that communicate with these neurons. The contribution of this slow depolarization is evident in the recording shown in Fig. 6. In this recording a series of depolarizing current pulses injected into B63 led to progressively more spikes in B63 and progressively larger depolarizations of B31/B32. After the third depolarization B63 began to fire even in the absence of stimulation, and after the fourth depolarization a buccal motor program was elicited. Figure 6 illustrates only the start of this program. Note that B31/B32 remained somewhat depolarized between pulses delivered into B63. This depolarization may be attributed to a slow EPSP between B63 and B31/B32, in addition to the fast EPSP already documented above, or to the activation of a slow, voltage-dependent process in B63, B31/B32, or additional coupled neurons. Experiments to determine the nature of this process were not performed.


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FIG. 6. Effect of 4 successive depolarizing pulses injected into B63 neuron. Note that number of action potentials elicited by pulse progressively increases and degree of depolarization produced by these action potentials in contralateral B31/B32 neuron progressively becomes larger. Also note that B63 continues firing after 3rd pulse, even in absence of depolarization, and buccal motor program is initiated after 4th pulse. Note that figure illustrates only the start of this program.

NECESSITY OF B63 ACTIVITY FOR THE BUCCAL MOTOR PROGRAMS. To determine whether their activity is a necessary component of buccal motor programs, in four preparations individual B63 neurons were hyperpolarized and prevented from firing. In all preparations, removal of B63 was found to change significantly either the presence or form of programs.

In one experiment (Fig. 7), buccal motor programs were elicited by sustained depolarization of a cerebral ganglion commandlike neuron, CBI-2. Programs were monitored by intracellular recordings from a B31/B32 and a B63 neuron, via extracellular recordings from the radula nerve, and by phasic activity in CBI-2, which becomes hyperpolarized during the retraction phase of a buccal motor program. B63 was hyperpolarized during this activity to prevent its firing. This blocked the expression of buccal motor program for as long as the hyperpolarization was maintained, and bursting was restored after the hyperpolarization was terminated.


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FIG. 7. Effect of hyperpolarizing B63 neuron during buccal motor programs elicited by CBI-2. Series of buccal motor programs were elicited by depolarizing commandlike cerebral ganglion neuron CBI-2. Programs were monitored by recording extracellular activity from radula nerve as well as intracellular activity from B31/B32 neuron, B63, and CBI-2. Note that CBI-2 is phasically hyperpolarized along with B31/B32 and B63. Hyperpolarizing B63 (down-arrow  up-arrow ) and preventing it from firing blocks expression of buccal motor program in other cells. However, note that some weak activity is recorded in nerve when program would have occurred.

In another experiment, B63 was repeatedly hyperpolarized during the protraction phase of buccal motor programs that had already begun, either spontaneously or via stimulation of B63. In all cases, hyperpolarizing B63 did not immediately affect either the ipsilateral or the contralateral B31/B32, indicating that after it is initiated, the sustained depolarization can be maintained for some period in the absence of firing in the contralateral B63 (Fig. 8). This finding supports the hypothesis that summation and facilitation of fast EPSPs from B63 to the contralateral B31/B32 is not exclusively responsible for the sustained depolarization. A slow EPSP or a slow, voltage-dependent process contributes to the sustained depolarization. However, in all cases in which B63 was hyperpolarized the sustained depolarization in the contralateral B31/B32 neurons stopped after a delay of ~1 s, indicating that the maintenance of the sustained depolarization in the contralateral B31/B32 is nonetheless strongly dependent on depolarization and firing in B63 (Fig. 8,A-C).


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FIG. 8. Effects of hyperpolarizing single B63 neuron during buccal motor program. Note that all 3 examples are from same preparation, indicating that different effects observed do not arise from variability between preparations. A: buccal motor program was initiated by depolarizing B63 (current injections are shown below B63 trace). Program was monitored by recording from both ipsilateral and contralateral B31/B32 neuron. One second after B63 stopped firing there was cessation of sustained depolarization in contralateral B31/B32 neuron. Release of B63 hyperpolarization restored activity to contralateral B31/B32 neuron and also excited ipsilateral B31/B32 neuron. Sustained depolarization in both was terminated by large hyperpolarization, which reflects start of retraction phase of buccal motor program. B: 2nd example in which B63 was depolarized and then hyperpolarized to prevent firing. In this example, after a delay of ~1 s, hyperpolarization of B63 terminated buccal motor program. Sustained depolarization in both ipsilateral and contralateral B31/B32 neurons stopped, and there was no subsequent retraction phase (note absence of hyperpolarization in all 3 traces). Immediately after interrupted program, 2nd buccal motor program was initiated, but this was allowed to proceed without interruption. Note difference in appearance of the 2 programs. C: B63 was hyperpolarized during spontaneous buccal motor program. This inhibited activity in both ipsilateral and contralateral B31/B32 neurons but did not prevent subsequent appearance of retraction phase of buccal motor program, as monitored by large hyperpolarization seen in all 3 neurons (note that start of hyperpolarization is reversed in B63).

Although the effects of hyperpolarizing B63 on the contralateral B31/B32 neurons were quite consistent, the effects on the ipsilateral B31/B32 were somewhat variable. In some runs, hyperpolarizing B63 did not block the sustained depolarization in the ipsilateral B31/B32 neuron (Fig. 8A). Note that in the example shown, release of the B63 hyperpolarization reinstated firing in B63, and this led to a restoration of activity to the contralateral B31/B32 neuron and also excited the ipsilateral B31/B32 neuron. The sustained depolarization in both B31/B32 neurons and in B63 was terminated by the large spontaneous hyperpolarization that marks the start of the retraction phase of a buccal motor program in these neurons. By contrast, in other runs (Fig. 8, B, 1st burst, and C), preventing B63 from firing stopped the sustained depolarization in both the ipsilateral and contralateral B31/B32 neurons after a delay of 1-2 s. In some cases (Fig. 8B) there was no subsequent retraction phase, as monitored by the absence of the large hyperpolarization in all three neurons (compare the cessation of the sustained depolarization in Fig. 8B, 1st and 2nd bursts). In other cases, the retraction phase was initiated even though activity representing protraction was eliminated in all of the three neurons that were recorded (Fig. 8C) (note that the start of the retraction phase IPSP causes a depolarization in B63, because the cell is hyperpolarized below the reversal potential of the IPSP). These data show that retraction can be initiated even when B63 and the B31/B32 neurons have been silenced.

B34 excites CPG elements and drives buccal motor programs

In addition to B63, a second neuron was found that excites B31/B32. This neuron, B34, was described previously as being adjacent to B31/B32 (Susswein and Byrne 1988), and was said to be similar to B31/B32 in that no spikes were ever recorded from its soma. B34 was described as being weakly coupled to B31/B32, and it displayed a small depolarization in phase with the large sustained depolarization in B31/B32. In the present study, slow depolarization and the absence of spiking in B34 was recorded during many spontaneous (Fig. 9A) and elicited buccal motor programs. Such recordings also showed that the slow depolarization in B34 is terminated by a large IPSP that is in phase with the IPSP in B31/B32 that terminates the sustained depolarization and that marks the retraction phase in these neurons. This IPSP is due to the effect of B64 on both B31/B32 and B34 (Hurwitz and Susswein 1996).


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FIG. 9. Properties of B34. A: in many buccal motor programs B34 was depolarized in phase with B31/B32, but did not fire. B: in some buccal motor programs B34 fires toward end of sustained depolarization of B31/B32, e.g., toward end of protraction phase of program. C: depolarization of B34 (up-arrow  down-arrow ) elicited action potentials only after a delay of ~2 s. These action potentials elicited 1-for-1 facilitating EPSPs in contralateral B31/B32 neuron.

In addition to B34 activity similar to that previously described, we have now found that in some programs B34 sustains action potentials. These are usually seen after the soma has been depolarized for >= 1 s (Fig. 9, B and C). Thus B34 differs from B63 in that it does not fire during every cycle of buccal motor programs. In addition, it often fires only at the end of the sustained depolarization of B31/B32 rather than throughout the depolarization.

CONNECTIONS FROM B34 TO B31/B32 AND TO ITS CONTRALATERAL HOMOLOGUE. Action potentials in B34 elicit one-for-one EPSPs in the contralateral B31/B32 neurons (Fig. 9C) without affecting the ipsilateral B31/B32 neurons. These EPSPs are still observed in 3 × Ca2+, 3 × Mg2+ seawater, indicating that the connection is monosynaptic.

Injecting brief depolarizing pulses into a B34 neuron during recording from the contralateral B34 reveals that spikes induce one-for-one, seemingly monosynaptic EPSPs in the contralateral B34 neuron (Fig. 10A). Injections of hyperpolarizing current reveal that the B34 neurons are also weakly electrically coupled (Fig. 10B).


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FIG. 10. B34 excites and is coupled to its contralateral homologue. A: brief depolarizations of B34 neuron caused spikes, which were followed 1-for-1 by EPSPs in contralateral B34 neuron. Note that EPSPs ride on slow coupling potential. B: brief hyperpolarizations of B34 neuron cause contralateral B34 neuron to be hyperpolarized as well, indicating that the 2 B34 neurons are electrically coupled.

DEPOLARIZATION OF B34 ELICITS FIRING AFTER A LONG DELAY. The response of B34 to a sustained depolarization is reminiscent of that seen in neurons having a transient potassium current (A current). In such neurons depolarization activates a K+ current that stabilizes the membrane and prevents firing. The neurons begin firing only after the K+ current inactivates (Levitan and Kaczmarek 1991). In addition, repeated brief depolarizations cause progressively more spiking. This could be explained by a process such as the inactivation of an A current by the earlier depolarizations. We did not explore the ionic basis of the delayed firing in B34. However, we did examine whether repeated depolarizations cause an increase in B34 firing, as would be caused by an inactivating outward current.

Repeated depolarizations of B34 significantly and progressively increased the number of action potentials elicited in response to the stimulus. Such stimuli also revealed a prominent growth in the amplitude of the EPSPs recorded in B31/B32 (Fig. 11A). Thus repeated stimulation of B34 produced an increase in excitation of B31/B32 by two mechanisms: 1) an increase in the number of action potentials elicited by a pulse, which would lead to summation of the EPSPs elicited in B31/B32, and 2) an increase in the amplitude of the EPSPs elicited by the action potentials.

We quantified the amplification of the depolarization seen in B31/B32 as a result of repeated depolarizing pulses delivered to B34. Twelve runs were performed in four animals (Fig. 11B). In this experiment B34 neurons were given a series of identical depolarizing pulses. After the appearance of the first action potential in B34, an additional four pulses were delivered, for a total of five pulses with B34 action potentials (Fig. 11A). The number of spikes elicited by each pulse was measured, as were the amplitudes of the EPSPs elicited in B31/B32 as measured from the start of each EPSP to the peak depolarization (Fig. 11B). Amplitudes were normalized to the amplitude of the first EPSP elicited. Both the number of spikes and the amplitude of the depolarizations produced by these spikes increased from the first to the fifth stimulus. The first stimulus regularly and reliably elicited a single action potential, whereas the fifth pulse elicited from five to nine action potentials. In addition, successive EPSPs elicited within a stimulus became progressively larger. In the fifth stimulus, EPSPs depolarized B31/B32 by a mean of 4 times more than did the EPSP elicited during the first stimulus, and some EPSPs depolarized B31/B32 by up to 10 times more than did the first EPSP. In addition to displaying facilitation during a train of stimuli, there was also some facilitation seen across trains. This was measured by comparing the first EPSP elicited by each of the five stimuli. Although the first EPSP remained relatively constant through the first three stimuli, the first EPSP elicited by the fourth stimulus was 32.5 ± 16.5% (mean ± SE) larger than the EPSP elicited by the first stimulus and the first EPSP elicited by the fifth stimulus was 63 ± 23% larger. These data show that both summation and facilitation are likely to contribute to the amplification of the excitation of B31/B32 by B34.

We explored whether growth in the amplitude the EPSPs could depolarize B31/B32 sufficiently that a buccal motor program is elicited by firing B34 (Fig. 12). B34 was stimulated with sets of 10 constant current depolarizations every 5 s. The depolarizations all elicited single action potentials during the first three sets of stimuli. Growth in the amplitude of the EPSPs in B31/B32 was seen in the first set of stimuli, and this growth was increased in the second set. Further growth in the EPSP amplitude during the third set caused a sufficient depolarization in B31/B32 to induce a buccal motor program. However, the fourth set of stimuli did not induce action potentials in B34, presumably because it occurred while B34 was hyperpolarized by the large IPSP elicited by B64 that is an indication of the retraction phase of a buccal motor program. The next set of stimuli elicited facilitation comparable with that in the first set.


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FIG. 12. B34 can initiate buccal motor programs. B34 was stimulated with 10 constant current depolarizations every 5 s (horizontal bars underneath B34 trace). First 3 stimuli elicited action potentials in B34 and gradually increasing depolarizations of B31/B32. Depolarization elicited by 3rd stimulus induced buccal motor program. Fourth stimulus did not induce action potentials in B34, whereas 5th stimulus elicited facilitation comparable with that produced in 1st stimulus.

The properties of B34 were explored further by injecting a series of progressively larger depolarizing currents into the cell (Fig. 13, A, 1st 4 traces, and B). Larger pulses caused a two-component response. An initial fast component was followed by a slower, more sustained component. The later component presumably reflects the activation of a slow outward current that stabilizes the membrane potential in response to depolarizing currents. A longer current pulse causes inactivation of this current and produces sufficient depolarization of B34 so that it begins to fire (Fig. 13A, last trace). These data showed that B34 behaves as if it fires after a voltage-dependent outward current is inactivated. In many buccal motor programs B34 activity is seen after a delay, toward the end of the protraction phase of the burst. However, repeated depolarizing pulses to B34 could cause it fire earlier. One would predict that B34 activity would be seen during a considerable portion of the protraction phase if a program has been preceded by previous bursts in which B34 was already depolarized. We found this to be the case (not shown).


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FIG. 13. Inactivation of outward current underlies delay in B34 activity. A: 1st 4 sets of traces show series of progressively larger current injections (bottom trace) into B34 and voltage changes elicited by current (top trace). Larger current pulses elicited response with 2 phases, with later phase showing marked rectification. Fifth set of traces: effect of current pulse that is maintained for longer period. Action potentials are eventually elicited after delay. Note that amplitude of current injected in last set of traces is same as in preceding briefer current injection. B: plot of voltage elicited by 1st 4 current injections shown in A. Open circles: amplitude of 1st phase. Filled squares: amplitude of 2nd phase.

Connections to additional buccal ganglia neurons reveal differences and similarities in B63 and B34 function

INTERCONNECTIONS BETWEEN B63 AND B34. B63 and B34 are mutually excitatory. However, the excitation of B63 produced by firing B34 is stronger than is the excitation of B34 produced by firing B63. Thus depolarization and firing of B34 causes vigorous firing in B63, whereas depolarization and firing of B63 causes a weak depolarization in B34 similar to that seen in buccal motor program in which B34 is not active (not shown). Additional recordings showed that spikes in B34 cause one-for-one, seemingly monosynaptic EPSPs in B63 (Fig. 14A), whereas spikes in B63 do not cause one-for-one EPSPs in B34.


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FIG. 14. Connections from B63 and B34 onto B63 and B64. A: B34 causes 1-for-1 EPSP in B63. B: firing B63 and B34 both produce inhibition of B64. Effect of B34 is larger than that of B63. C: inhibitory postsynaptic potentials (IPSPs) from both B63 and B34 are retained in high-divalent-cation solution, indicating that connections are monosynaptic.

MUTUAL INHIBITION BETWEEN PROTRACTOR AND RETRACTOR CPG ELEMENTS. A previous study identified B64, a CPG element that is partially responsible for the phase switch from protraction to retraction (Hurwitz and Susswein 1996). B64 inhibits neurons that are active during protraction (including B31/B32, B63, and B34) (see Hurwitz and Susswein 1996) and excites many neurons that are active during retraction (see Fig. 2). Both B63 and B34 were found to inhibit B64, but the inhibition from B34 was much more powerful than that from B63 (Fig. 14B). IPSPs from B63 and B34 are still seen in 3 × Ca2+, 3 × Mg2+ seawater (Fig. 14C), indicating that they are likely to be monosynaptic. The IPSPs produced by B63 were previously observed in B64 during spontaneous bursts of activity in B31/B32 and followed one-for-one the EPSPs in B31/B32 that are caused by B63 (Hurwitz and Susswein 1996).

In addition to inhibiting B64, B63 and B34 inhibited many of the neurons in the ventral cluster of the buccal ganglia that are inhibitory followers of identified neurons B4 and B5. Many of these neurons have been shown to be motor neurons to various buccal muscles, and they generally fire during the retraction phase of buccal programs. The effects of B34 were generally stronger than were those of B63. Surprisingly, neither B63 nor B34 affected the B4/B5 neurons.

EXCITATION OF ADDITIONAL PROTRACTOR MOTOR NEURONS. B61 and B62 are motor neurons innervating the I2 muscle of the buccal mass, which is responsible for a major portion of the radula protraction (Hurwitz et al. 1996). The I2 muscle is also innervated by the B31/B32 neurons. However, B31/B32 function as both CPG elements and motor neurons, whereas B61/B62 are conventional motor neurons (Hurwitz et al. 1994). In addition, B31/B32 fire during the protraction phase of every buccal motor program, whereas B61/B62 fire during some cycles of buccal motor programs (Hurwitz et al. 1996). The synaptic relationships between B34 and B63 and B61/B62 were examined in five preparations.

B34 is electrically coupled to the contralateral B61/B62 neurons, whereas B63 is not (Fig. 15, A and B). Both B63 and B34 produce one-for-one EPSPs in the contralateral B61/B62 neurons (Fig. 15, C and D). The EPSPs from B63 are still seen in 3 × Ca2+, 3 × Mg2+ seawater (Fig. 15C), indicating that they are monosynaptic. The connections from B34 to B61/B62 and to B31/B32 were compared (Fig. 15D). These connections were tested in 0.1 × Ca2+, 4 × Mg2+ seawater to preserve the facilitation produced by B34 in B31/B32. In this solution one-for-one EPSPs were produced by B34 in both B31/B32 and in B61/B62. However, the EPSPs in B31/B32 showed strong facilitation, whereas those recorded in B61/B62 did not.


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FIG. 15. Connections to B61/B62. A and B: B34, but not B63, is electrically coupled to B61. C and D: B63 and B34 elicit monosynaptic EPSPs in B61. Note that EPSPs were still seen in solutions that either block polysynaptic pathways (C) or reduce efficacy of chemical synaptic transmission (D). Also note that EPSPs produced by B34 in B61 do not show the strong facilitation shown by synchronous EPSPs in B31.

Although both B63 and B34 excited B61/B62, their effects were quite different. In programs in which B34 is not active, B61/B62 typically responded with few action potentials. By contrast, when B34 is active, robust firing in B61/B62 was seen (Fig. 16), showing that the variability in the likelihood of B61/B62 and of B34 being active in a buccal motor program is related.


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FIG. 16. B34 and B61/B62 activity are correlated. A: buccal motor programs were elicited by depolarization of B31 (up-arrow  down-arrow ). This caused depolarization of B34 and B61 in phase with B31 depolarization, but neither B34 nor B61 fired. Depolarizations in all 3 neurons was terminated by hyperpolarizations that are attributed to activation of B64. B: buccal motor programs were elicited by depolarization of B34. In these programs there was prominent firing in both B34 and in B61 in phase with depolarization of B31.

Because B34 is likely to fire after it has been depolarized for some time, one might predict that B61/B62 is also likely to fire late in the protraction phase. In addition, one might predict that B61/B62 should fire preferentially during buccal motor programs with a relatively long protraction phase. These predictions were confirmed (Fig. 17A). However, it is important to note that activity in B61/B62 often preceded firing in B34, indicating that both neurons are likely to be driven in tandem by a common excitatory input that causes the protraction phase of a buccal motor program to be lengthened.


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FIG. 17. Events driving B34 also drive B61/B62. A: 2 spontaneous buccal motor programs from same preparation. Both B34 and B61 have a tendency to fire late in protraction phase. In buccal motor programs with a long protraction phase there is an increase in activity of both B34 and B61/B62. B: buccal motor programs were elicited by intracellular stimulation (up-arrow  down-arrow ) of B63 neuron. B34 was depolarized and caused to fire twice (up-arrow down-arrow down-arrow up-arrow ), once before and once during protraction phase of buccal motor program. First stimulation of B34 caused depolarization and weak firing in B61/B62. Second stimulation elicited vigorous activity in B61/B62.

Depolarizing B34 and causing it to fire during a burst in which it would have been silent led to increased firing of B61/B62 (Fig. 17B). In the example shown, firing B34 in the absence of a burst (1st depolarization of B34) led to depolarization and a single action potential in B61/B62. By contrast, a similar stimulus to B34 during the protraction phase of a burst (2nd depolarization of B34) caused brisk activity in B61/B62. During bursts in which B34 was not active, the activity in B61/B62 was considerably weaker. These data show that B61/B62 and B34 are driven by common activity of an unknown source, which makes it likely that both will fire. In addition, activity in B34 augments the excitation onto B61/B62, increasing its firing.

B63 AND B34 DIFFERENTIALLY AFFECT B8, A MONITOR OF REJECTION-LIKE PROGRAMS. The data above indicate that B34 activity is correlated with, and is partially responsible for, activity in the B61/B62 motor neurons during the protraction phase of a buccal motor program. Because B61/B62 innervates the I2 muscle, activity in B61/B62 is likely to cause an increase in the amplitude of protraction movements. During rejection movements, the protraction phase of a buccal motor program is the power phase, whereas during ingestion, retraction is the power phase. The effect of B34 on B61/B62 suggests that buccal motor programs in which B34 is active represent rejection movements, whereas programs in which B34 is silent represent ingestion. We tested this possibility by examining the effects of B63 and B34 on the B8 motor neurons, which innervate muscle I4. This muscle is partially responsible for closing the radula. During rejection movements the B8 neurons are active during protraction, but during ingestion movements they are active during retraction (Morton and Chiel 1993b). If activity in B34 is related to rejection, one would predict that B34 would excite B8. By contrast, because the B31/B32-B63 complex is active during both ingestion and egestion movements (Hurwitz et al. 1996), one would predict that B63 would not excite the B8 neurons. We tested these predictions.

B63 produced one-for-one IPSPs in the contralateral B8 that were still present in 3 × Ca2+, 3 × Mg2+ seawater (Fig. 18A), indicating that the connection is probably monosynaptic. Initiating a buccal motor program by depolarizing B63 led to a mixture of EPSPs and IPSPs in the contralateral B8 during the protraction phase, with a barrage of EPSPs in B8 during the retraction phase, when B63 is silent (not shown). Activity of B8 during retraction is indicative of an ingestion-like buccal motor program (Morton and Chiel 1993b).


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FIG. 18. Connections from B63 and B34 to B8. A: depolarization (up-arrow  down-arrow ) and firing B63 neuron produces 1-for-1 IPSPs in B8 in presence of 3 × Ca2+, 3 × Mg2+ solution, indicating that connection is monosynaptic. B: depolarization (up-arrow  down-arrow ) and firing B34 neuron produces 1-for-1 IPSPs in B8, followed by slow excitation and B8 firing. C: B34 was depolarized (up-arrow  down-arrow ) in 3 × Ca2+, 3 × Mg2+ seawater while hyperpolarizing constant current pulses were injected into B8 (horizontal bars). Firing B34 caused slow depolarization of B8 that was accompanied by conductance decrease. D: buccal motor programs were driven by depolarization of right B31/B32 neuron (horizontal bars underneath B34 trace mark times that B31/B32 was depolarized, but B31/B32 trace is not shown) during recording from the 3 neurons shown. Activity in B34 is associated with activity in B8 during protraction phase of buccal motor program. When B34 neuron was hyperpolarized and prevented from firing (down-arrow  up-arrow ), activity in contralateral B8 neuron was reduced.

B34 had a mixed effect on B8. B34 produced one-for-one fast IPSPs similar to those produced by B63, but these were followed by a slow excitation that led to B8 firing (Fig. 18B). The slow excitation was still maintained in 3 × Ca2+, 3 × Mg2+ seawater, indicating that it is also likely to be produced by a direct connection from B34 to B8 (Fig. 18C).

In some Aplysia synapses, a slow depolarization similar to that from B34 to B8 is caused by a decrease in K+ conductance (Byrne et al. 1979; Weiss et al. 1986b). We examined whether such a mechanism could account for the excitation of B8 by B34 by passing hyperpolarizing current pulses into B8 while B34 was stimulated. The amplitude of the response to current pulses increased during the slow excitation (Fig. 18C), suggesting that a decrease in conductance may accompany the excitation of B34.

If buccal motor programs in which B34 is active represent rejection responses, such programs should be characterized by activity in B8 during the protraction phase of a buccal motor program. In >10 preparations, we found that activity in B34 during the protraction phase was indeed correlated with activity in B8 during this phase (Fig. 18D), providing additional evidence that B34 activity represents a rejection-like buccal motor program. In addition, hyperpolarization of B34 to prevent it from firing strongly reduced the firing in B8 during protraction (Fig. 18D), indicating that B34 plays a role in driving B8 activity during the protraction phase of rejection-like programs.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

We have characterized two neurons, B63 and B34, that have access to a CPG in the Aplysia buccal ganglia controlling the consummatory phase of feeding. These neurons have many properties in common. Both are active during the protraction phase of buccal motor programs, and both have axons crossing into the contralateral buccal ganglion, where they excite the contralateral B31/B32 neurons and then exit the buccal ganglia via the contralateral cerebrobuccal connective. B63 and B34 are also both multiaction neurons having excitatory and inhibitory synaptic actions on different follower cells. B63 and B34 monosynaptically excite neurons active during protraction and inhibit B64 and other neurons active during retraction.

Despite these similarities, B63 and B34 differ greatly in their firing properties. B63 is easily fired and is active during all buccal motor programs that have been observed. Thus it is likely to have an integral function in organizing and initiating both ingestion and egestion movements. By contrast, B34 fires only after it has been depolarized for a considerable period of time, or after a number of shorter depolarizations, and therefore it is active only during some motor programs. Its function is thus more circumscribed than that of B63. The connections of B34 to other buccal ganglia neurons suggest that programs in which B34 is active may be rejection like and that B34 may have a role in generating such programs.

Reciprocal inhibition of protractor and retractor phase neurons

A summary of the synaptic connections between neurons discussed in the present report is shown in Fig. 19A. The diagram shows extensive chemical and electrical connections between neurons active during the protraction phase of buccal motor programs. Our data also indicate that interneurons that are active in the protraction and retraction phases of buccal motor programs are generally mutually inhibitory. B63 and B34 inhibit B64, which has a major role in initiating and maintaining the retraction phase, and also inhibit motor neurons that are active during the retraction phase. B64 in turn inhibits B31/B32, B34 and B63, and B61/B62, and excites many of the motor neurons that are active during the retraction phase.

Homology to other molluscan systems

Possible homologues of either B63 or B34 have been found in a number of other gastropod mollusks. In Pleurobranchaea, Clione, and Lymnaea, neurons have been found in the lateral portion of the buccal ganglia that are active during the protraction phase of buccal motor programs and that have axons exiting the buccal ganglia exclusively via the contralateral cerebral-buccal connective (Arshavsky et al. 1989b; Elliott and Benjamin 1985; Kovac et al. 1986). These interneurons can drive patterned bursts in the buccal ganglia in Clione and Lymnaea (Arshavsky et al. 1989b; Elliott and Benjamin 1985). In Pleurobranchaea these neurons excite commandlike neurons in the cerebral ganglion (Kovac et al. 1986). The number of these neurons found differs in the various gastropod species. One such neuron, termed the PIN1, is found in each buccal hemiganglion of Clione (Arshavsky et al. 1989b), whereas two such neurons (termed the CCD neurons) have been described in each hemiganglion in Pleurobranchaea (Kovac et al. 1986). Approximately 10 such neurons (termed N1 or N1M) (see Yeoman et al. 1995) are found in Lymnaea. In each preparation, these neurons act together and display ipsilateral as well as contralateral electrical coupling. In Clione, the PIN1 neuron apparently has an endogenous plateau potential (Arshavsky et al. 1989b). In none of the preparations do the relevant neurons display the delay in firing characteristicof B34.

Possible function of B63

INITIATION OF PROTRACTION. The bilaterally symmetrical B31, B32, and B63 neurons form a single functional unit. These six neurons are mutually excitatory, and sufficient excitation of any of them activates them all. The B31/B32-B63 complex is likely to play a critical role in the initiation and maintenance of the protraction phase of a variety of buccal movements. Chronic recordings have shown that the B31/B32 neurons are active during the protraction phase of biting, swallowing, and rejection movements (Hurwitz et al. 1996). The complex is directly excited by higher-order commandlike neurons (the cerebrobuccal interneurons) (see Rosen et al. 1991a) that initiate feeding (Hurwitz and Kupfermann, unpublished data). Firing in the complex leads to excitation of additional neurons active during protraction and contraction of the I2 muscle (Hurwitz et al. 1994, 1996) and also causes inhibition of neurons active during retraction (Figs. 14 and 18B).

Activation of the B31/B32-B63 complex also activates neuron B33, which is strongly coupled to B31/B32 (Susswein and Byrne 1988) and which may also be regarded as a member of the complex. B33 has a peripheral axon in the ipsilateral buccal nerve 3 (unpublished observations).

Groups of neurons active together and forming a single functional unit are also found in CPGs in other animals (Getting 1989; Pearson 1993; Selverston and Moulins 1985) as well as in the CPG controlling respiratory pumping in Aplysia (Byrne 1983; Koester 1989). However, in most other systems the component neurons acting together seem to be more similar in their physiological properties than are B63, B31/B32, and B33.

BILATERAL COORDINATION. The B31/B32-B63 complex contributes to the bilateral coordination of activity in the two buccal hemiganglia. Consummatory feeding movements in Aplysia differ from many other repetitive movements studied (Pearson 1993) in that activity is synchronous bilaterally, rather than occurring 90° out of phase bilaterally. Buccal motor programs are also bilaterally synchronous. However, early studies found that certain multiaction neurons have exclusively ipsilateral connections (Gardner 1977). In addition, many motor neurons produce exclusively ipsilateral contraction (Church and Lloyd 1991; Cohen et al. 1978; Rosen et al. 1982), although some motor neurons have bilateral effects (Church and Lloyd 1991; Lotshaw and Lloyd 1990). More recent studies have shown that contralateral or bilateral connections are common in identified CPG elements, thereby explaining the bilateral synchrony of activity seen in consummatory movements. Thus B31/B32 innervate the contralateral I2 muscle, whereas interneurons B51 (Plummer and Kirk 1990) and B64 (Hurwitz and Susswein 1996) have bilateral effects. We have now found that B34 has exclusively contralateral connections and that B63 produces mixed chemical and electrical EPSPs in the contralateral B31/B32 neurons and is electrically coupled to both the ipsilateral and contralateral B31/B32 neurons. These connections are likely to contribute to the strong synchrony of activity seen in the two buccal hemiganglia (Fig. 19B).

CONTRIBUTION OF B63 TO BUCCAL MOTOR PROGRAMS. We tested whether B63 neurons contribute to buccal motor programs by hyperpolarizing the neurons during programs. In addition to silencing the neuron, hyperpolarization revealed the presence of slow depolarizing potentials in B31/B32 that apparently contribute to B63 activity. In all cases silencing a single B63 neuron had an effect on buccal motor programs. In programs elicited by depolarizing and firing commandlike neuron CBI-2, hyperpolarizing B63 blocked the expression of programs (Fig. 7). When B63 was hyperpolarized after the start of the protraction phase (Fig. 8), there was no immediate effect on either the ipsilateral or the contralateral B31/B32 neurons. However, ~1 s later, activity in the contralateral B31/B32 neurons ceased. Activity in the ipsilateral B31/B32 sometimes also ceased, and the buccal motor program was sometimes also terminated. These experiments indicate that B63 has a role in the maintenance of the sustained depolarization in the contralateral B31/B32 neurons. In addition, B63 contributes to the maintenance of a buccal motor program.

Possible function of B34

B34 differs from the neurons in the B31/B32-B63 complex in that it fires only during rejection-like buccal motor programs. In addition, depolarizing B34 can initiate such programs.

B34 AMPLIFIES THE PROTRACTION PHASE OF A BUCCAL MOTOR PROGRAM. B34 activity is associated with buccal motor programs in which the protraction phase is relatively long and motor neurons B31/B32 and B61/B62 are strongly activated (Figs. 9 and 16). B34 excites the B31/B32 and B61/B62 motor neurons (Figs. 9 and 15), contributing to their activity during the protraction phase. Both B31/B32 and B61/B62 innervate the I2 muscle, a major contributor to protraction movements (Hurwitz et al. 1996). Chronic electromyographic recordings from the I2 muscle during movement (see Figs. 10-12 in Hurwitz et al. 1996) have shown that relatively little activity that can be attributed to B61/B62 is recorded during biting or swallowing, but B61/B62 may play a strong role in rejection movements. During ingestion, the retraction phase of a feeding cycle is the power phase, in which food is moved, whereas during rejection, protraction is the power phase. A rejection movement requires a larger, more powerful protraction phase than that seen during ingestion movements. Enhanced activity in the B31/B32 and B61/B62 motor neurons when B34 is active would contribute to the amplification of the protraction phase during rejection.

B34 CONTRIBUTES TO RADULA CLOSING DURING PROTRACTION. B34 activity is correlated with activity of motor neuron B8 during the protraction phase of a buccal motor program (Fig. 18). B8 innervates the I4 closer muscle. Previous work (Morton and Chiel 1993a) has shown that radula closing accompanies the retraction phase of ingestion movements, but occurs along with protraction during rejection. This is associated with a phase switch of the activity in B8 (Morton and Chiel 1993b). During ingestion movements B8 is active in phase with retraction, whereas during rejection B8 fires during protraction. B34 has mixed but primarily excitatory effects on B8, thereby making it more likely to fire during the protraction phase (Fig. 18). However, it is important to note that other neurons are likely to act in tandem with B34. Thus in some buccal motor programs B61/B62 or B8 began to fire before B34 became active, indicating that other neurons also drive these cells (Fig. 17). The decreased-conductance EPSP caused by B34 onto B8 may amplify the effects of additional neurons that excite B34 during the protraction phase. The ability of a conductance-decrease EPSP to amplify the effects of other inputs has been shown in the motor system controlling inking in Aplysia (Byrne et al. 1979).

B34 BEHAVES AS THOUGH IT HAS A SLOWLY INACTIVATING OUTWARD CURRENT. B34 becomes active and fires after a delay. We have not investigated the biophysical basis of the delayed response. However, similar responses are seen in the L14 neurons of the Aplysia abdominal ganglion (Byrne 1982; Shapiro et al. 1979), in the VSI-B neurons in the Tritonia swimming CPG (Getting 1983), and in other molluscan neurons (Connor and Stevens 1971).

The presence of an inactivating outward current in B34 may contribute to the ability of this neuron to control ingestion versus egestion-like buccal motor programs. During ingestion, the current may be prominent, and its presence will prevent B34 from firing. By contrast, during rejection the current may inactivate, allowing B34 to fire. Recent studies (I. Hurwitz and K. R. Weiss, unpublished data) have shown that B34 synthesizes a number of peptide cotransmitters. Release of these transmitters could contribute to the rewiring of the CPG, so that it becomes more appropriate for generating rejection.

POSSIBLE ROLE OF B34 IN PLASTICITY OF FEEDING. Feeding in Aplysia can be modified by a variety of interesting processes (Botzer et al. 1991; Hurwitz and Susswein 1992; Kupfermann et al. 1991; Schwarz et al. 1988; Susswein et al. 1986). These processes affect both appetitive and consummatory components of feeding (Kupfermann et al. 1991; Schwarz et al. 1988). Effects on consummatory movements include changes in the probability of eliciting a rejection responses (Schwarz et al. 1988) as well as changes in the amplitude and/or frequency of ingestion responses (Susswein et al. 1976). Access to B34 is of particular interest because it may be affected by processes altering the probability to respond with rejection in place of ingestion.

    ACKNOWLEDGEMENTS

  We thank S. Markovich for help in preparing the figures and H. Chiel for comments on the manuscript.

  This work was supported by U.S.-Israel Binational Science Foundation Grant 93-224 and National Institute of Mental Health Grant MH-35564. Aplysia were provided by the NCRR National Resource for Aplysia at the University of Miami under Division of Research Resources Grant RR-10294.

    FOOTNOTES

   Present address of I. Hurwitz: Dept. of Physiology, Box 1218, Mount Sinai School of Medicine, 1 Gustave Levy Place, New York, NY 10029.

  Address for reprint requests: A. J. Susswein, Dept. of Life Sciences, Bar Ilan University, Ramat Gan 52 900, Israel.

  Received 20 August 1996; accepted in final form 5 May 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society