Dopaminergic neuron B20 generates rhythmic neuronal activity in the feeding motor circuitry ofAplysia

226

Brain Research, 630 (1993) 226-237 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00

BRES 19498

Dopaminergic neuron B20 generates rhythmic neuronal activity in the feeding motor circuitry of Aplysia T h o m a s T e y k e * * , S t e v e n C. R o s e n , K l a u d i u s z R . W e i s s

* * *, I r v i n g K u p f e r m a n n

*

Center for Neurobiology and BehaL,ior, College of Physicians and Surgeons Columbia University, 722 West 168th S t r e e t - Research Annex, New York, NY, USA (Accepted 20 July 1993)

Key words: CPG; Command neuron; Feeding behavior; Dopamine

We have identified a buccal neuron (B20) that exhibits dopamine-like histofluorescence and that can drive a rhythmic motor program of the feeding motor circuitry of Aplysia. The cell fires vigorously during episodes of patterned buccal activity that occur spontaneously, or during buccal programs elicited by stimulation of identified cerebral command-like neurons for feeding motor programs. Preventing B20 from firing, or firing B20 at inappropriate times, can modify the program driven by the cerebral feeding command-like neuron CBI-2. When B20 is activated by means of constant depolarizing current it discharges in phasic bursts, and evokes a sustained coordinated rhythmic buccal motor program. This program incorporates numerous buccal and cerebral neurons associated with aspects of feeding responses. The B20-driven program can be reversibly blocked by the dopamine-antagonist ergonovine, suggesting that dopamine may be causally involved in the generation of the program. Although firing of B20 evokes phasic activity in cerebral command-like neurons, the presence of the cerebral ganglion is not necessary for B20 to drive the program. The data are consistent with the notion that dopaminergic neuron B20 is an element within the central pattern generator for motor programs associated with feeding.

INTRODUCTION Feeding behavior in Aplysia has provided an advantageous system for the study of the neural mechanisms underlying the modulation of behavior by motivational state ~9'2~'4°'46. The role of specific neural circuitry and of a variety of neuromodulatory substances has been studied 6"8'48. Several neurons have been described that are capable of generating the rhythmic buccal motor programs (BMPs) that underlie coordinated movements of the buccal mass, a complex muscular organ that executes biting, swallowing and rejection. A small number of identified neurons, located in the cerebral ganglion and projecting axons to the buccal ganglion (cerebral-to-buccal interneurons, or CBIs), reliably drive robust and sustained BMPs 36 that lead to concomitant movements of the buccal mass 34. There is also evidence that, under certain conditions, buccal programs can be elicited by the firing of neurons located in the buccal ganglion 19'31'4°. Rhythmic BMPs

can also be elicited by bath application of several transmitters to the isolated nervous systems of a number of molluscan species. In Aplysia 37, as well as in a number of other molluscan species, dopamine is capable of initiating or modulating feeding motor programs. The effects of dopamine have been reported for Lymnaea 22, Limax 49, and Helisoma 28"44. Furthermore, the neuropil of the buccal and cerebral ganglion contains extensive dopamine histofluorescence, and dopamine-containing cells have been described in the buccal and cerebral ganglia of Aplysia 14"15"32'45, and in other molluscs, including Lymnaea 1°, Helisoma 28'43, and Limax 3°. The most detailed description of dopaminergic neurons in Aplysia has been obtained using a modification of the formaldehyde-glutaraldehyde (FaGlu) method 1~. Five cells in the buccal ganglion, and about 70 cells in the cerebral ganglion exhibit dopamine-like histofluorescence, and 3 dopamine-positive axons usually are revealed in each cerebral-buccal connective ~4. Spectral analysis has in-

* Corresponding author. Fax: (1) (212) 960-2410. ** Current address: lnstitut fiir Zoologie (III) Biophysik, Johannes Gutenberg-Universit~it, Saarstr. 21, 6500 Mainz, Germany. * ** Current address: Dept. of Physiology and Biophysics, Mt. Sinai Medical Center, One Gustave Levy PI., New York, NY 10029, USA.

227 dicated that the fluorescence is due specifically to the presence of dopamine TM. The current experiments are part of our attempt to study the role of dopamine in feeding in Aplysia and to establish the neuronal basis of the dopamine driven motor program. To that end, we sought to identify dopaminergic neurons capable of driving BMPs. Utilizing the modified FaGlu technique, we first confirmed the location of putative dopaminergic neurons in the buccal and cerebral ganglia, and then employed electrophysiological techniques to examine their role in feeding. In a previous study we described a dopaminepositive cerebral cell (CBI-1) that is capable of eliciting a single cycle of buccal activity 37. In this p a p e r we present the results of our study of a pair of dopaminepositive neurons located on the rostral surface of the buccal ganglion. Our results indicate that these neurons can drive a sustained rhythmic program and are components of a buccal ganglion pattern generator associated with feeding responses. MATERIALS AND METHODS

Subjects Experiments were performed on Aplysia californica (250-400 g) obtained from Marinus, Inc. (Long Beach, CA). The animals were dissected following immobilization by injection of an isotonic MgCI2 solution (25% of body weight) into the haemocoel. Three types of preparations were used: (1) a preparation consisting only of the cerebral and buccal ganglion pinned to a sylgard-lined recording chamber (Sylgard 184; Dow Corning); (2) an isolated cerebral-buccal ganglia preparation, in which each ganglion was placed in a separate chamber permitting the cerebral-buccal connectives to pass through slots that were sealed with Vaseline; and (3) a reduced preparation in which either the head, the buccal mass, or the pharynx and anterior part of the esophagus were left attached to the ganglia. The ganglia were pinned in a separate subchamber within the recording chamber. The preparations were maintained in artificial seawater (Instant Ocean) at room temperature. The connective tissue sheathes of the ganglia were surgically removed. Electrophysiology Multiple channel intracellular recordings were carried out using thin walled, double-barreled glass microelectrodes. The recording electrode was filled with 2 M potassium acetate, and the stimulating electrode was filled with a dye that could be iontophoreticaUy ejected into the cell body. Rhodamine lissamine dye (3% aqueous solution) was used in combination with a subsequent histofluorescence dopamine assay (FaGlu). To determine the fine morphology of the neuron, electrophysiological identification was followed by injection of 5(6)-carboxyfluorescein dye (Kodak; 5% in 0.1 M potassium acetate). Processes at a long distance from the cell body could be visualized when the ganglia were maintained for 12-24 h at 5°C in the presence of probenecid (see ref. 36), which suppresses the extrusion of the dye 39. The electrodes were beveled to tip resistances of 15-30 M,O, and standard recording and data acquisition techniques were used. Cells were identified based on published criteria 6,7A2.

Morphology Following electrophysiological recordings, putative dopaminecontaining neurons were marked by iontophoretic ejection of rhodamine dye. The ganglia were then fixed for 3-10 h using a solution

of 4% paraformaldehyde and 0.6% glutaraldehyde (FaGlu), which causes catecholamine-containing neurons to fluoresce under UVlight TM. The ganglion wholemounts were viewed with a Leitz fluorescence microscope with filter pack 'D' (excitation filter: 355-425 nm; barrier filter: 460 nm), which causes catecholamine containing tissue to appear blue-green. Rhodamine lissamine filled cells were identified under fluorescent light with filter pack 'D2' (excitation filter: 530-569 nm; barrier filter: 580 nm). The nomenclature for buccal neurons and nerves follows that of Gardner ~2.

RESULTS

Application of dopamine to the cerebral, but not the buccal ganglion, evokes a BMP In a preparation of the cerebral and buccal ganglia of Aplysia, application of dopamine close to the cerebral ganglion was very effective in driving a BMP 3v. Reliable effects required high concentrations of dopamine, perhaps because of the presence of the sheath and possible combined inhbitory and excitatory actions (see discussion of Ascher t). To determine which ganglion the dopamine was acting on, the buccal and cerebral ganglia were placed in separate chambers with the intact connectives passing through Vaseline sealed passages. Application of dopamine to the cerebral ganglion alone was effective in eliciting a sustained rhythmic BMP (Fig. 1A), even when the dopamine was prevented from contacting the buccal ganglion. By contrast, perfusion of dopamine onto the buccal ganglion was never found to cause a rhythmic pattern even when a concentration of up to 10 -3 M was used (n = 10 preparations). At the higher concentrations, some motor neurons became intensely active but without any obvious signs of patterned activity. Fig. 1B shows that 10 -3 M dopamine applied to the isolated buccal ganglion excited some cells (C12, MN-1), inhibited others (MN-2) and had a mixed excitatory-inhibitory effect on others (B4). The continual presence of dopamine, rather than promoting rhythmic activity, may have evoked increased inhibitory activity (note the strong hyperpolarization of motor neuron MN-2, and the presence of a prolonged hyperpolarization following brief excitation of B4 in Fig. 1B). Based on the findings that dopamine activates a BMP only when applied to the cerebral ganglion, and that the buccalcerebral connectives contain dopamine-positive axons, we sought to determine if dopamine-containing buccal-cerebral interneurons might drive a rhythmic program. We indeed found in the buccal ganglion, a dopamine-containing neuron that was capable of driving a BMP. The neuron sends an axon to the cerebral ganglion, but surprisingly, it can evoke a BMP even when that axon is severed (see later section). Thus there a p p e a r to be two independent phenomena. An effect of dopamine on the cerebral ganglion, and an

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Fig. 1. Bath application of dopamine to the isolated cerebral ganglion drives a buccal motor program. The buccal and cerebral ganglia were contained in separate sealed compartments with the C-B connectives intact. Application of 10 -4 M dopamine in ASW to the cerebral ganglion (A) produced a rhythmic buccal motor program, monitored by neuron B4 and unidentified buccal motor neurons, MN-1 and MN-2. The motor program also caused phasic firing of cerebral lip motor neuron, C12. Following several washes with ASW, 10 -3 M dopamine (or lower concentrations) applied to the isolated buccal ganglion (B) did not evoke a rhythmic pattern. Dopamine strongly excited MN-1 and had mixed effects on MN-2, C12 and B4. Note the absence of rhythmic activity in C12, suggesting that the buccal ganglion under these conditions did not provide rhythmic output to the cerebral ganglion.

effect of a dopamine-containing neuron on the buccal ganglion. We have concentrated on analyzing the actions of the dopamine-containing neuron. Dopamine-positive neuron, B20, is a buccal-cerebral interneuron

Using the FaGlu method we confirmed earlier reports of the relative location and gross morphology of 5 dopamine-positive neurons in the buccal ganglion and dopamine-positive axons within each cerebral-buccal connective 14'32'45. The FaGlu method reveals only limited details about the morphology of the cells. Moreover, this method is incompatible with the routine labeling of the neurons following electrophysiological experiments, particularly if intense fluorescent dyes, such as Lucifer yellow or 5(6)-carboxyfluorescein are used (both dyes have emission spectra overlapping that of the catecholamine histofluorescence). For double-

labeling experiments, we therefore combined electrophysiological identification of the neurons with Rhodamine-lissamine labeling, and subsequent treatment with FaGlu for unambiguous identification of the dopamine-positive cells. Fig. 2A shows the medial portions of the rostral surfaces of the two buccal hemiganglia and the commissural region after treating the preparation with FaGlu. A pair of bipolar neurons that exhibit dopamine-like histofluorescence can be seen near the buccal commissure. As shown below, these neurons can be unambiguously identified according to several electrophysiological criteria. They have been designated B20 neurons. In Fig. 2B and 2C, the left B20 neuron is shown at higher magnification, and its oval shape and bipolar processes are visible. Fig. 2B, taken under viewing conditions appropriate for rhodamine fluorescence, shows the neuron that was identified electrophysiologically and injected with rhodamine dye. Fig. 2C shows the same region of the ganglion, after the filter was changed for observation of dopamine histofluorescence, demonstrating that the dopaminepositive neuron B20 is identical to the rhodamine labeled neuron shown in Fig. 2B. Independent of the double-labeling experiments, we established criteria that permitted unambiguous identification of neuron B20 even without FaGlu fixation. These included (details will be presented below): a characteristic position, size, and shape of the soma; bilateral processes that travel in the C - B connectives and that extend, at least partially, into buccal nerve 3; a phasic response to constant current stimulation; the ability to drive a rhythmic BMP; and, monosynaptic connections to identified motor neurons B8 and B16. A nondopaminergic cell that also appears to be capable of driving a BMP is located near B20. In many respects the non-dopaminergic cell appears to be the previously described neuron B174, although in the current study its position and axonal tree does not appear to correspond to that of the previously described B17. Since exploration of the ganglion has not revealed that there is more than one cell with properties similar to that of B17, we have tentatively assumed that the nondopaminergic neuron in the vicinity of B20 is B17. We have found that B17 can be distinguished from B20 on the basis of several criteria other than the presence of dopamine fluorescence following FaGlu. B17 is located more medially in the ganglion. Like B20, B17 sends processes to the C - B connectives (see below), but unlike B20, it makes a monosynaptic excitatory connection to the cerebral neuron C44. In addition, although not described in the previous study of B17, the medially directed (commissural) process of the non-

229

b

Fig. 2. Morphological identification of the putative dopaminergic neuron, B20. A: low power view of the rostrai surface of the buccal ganglion following FaGlu fixation under illumination appropriate for dopamine-histofluorescence. The buccal commissure and the medial regions of the two buccal hemiganglia can be seen. A bilateral pair of elongated neurons each with two axonai processes show intense blue-green histofluorescence. Calibration bar = 250 p,m. B: high power view of candidate B20 neuron (left neuron in A), which was marked with rhodamine-lissamine dye following electrophysiological identification. The cell showed rhodamine (red) fluorescence when viewed with appropriate filters. C: same cell as in (B) also exhibited dopamine-like histofluorescence when observed with a filter appropriate for dopamine histofluorescence. B and C: calibration bars = 75 p,m.

230 dopaminergic cell is thicker than the laterally directed process, whereas the processes of B20 are both relatively thin. The detailed morphology of B20, shown in Fig. 3, was revealed by identifying the cell electrophysiologically and afterwards injecting it with 5(6)-carboxyfluorescein. Fluorescence microscopy of the unfixed ganglion revealed that the ipsi- and contralateral axons of B20 branch extensively in the buccal ganglion. The two main branches traverse the neuropilar region of the two hemiganglia. Both branches bifurcate at lateral positions, one branch projecting into the C - B connective, the other projecting, at least part way, into buccal nerve 3. When the ganglion was maintained in probenecid for 24 h the processes of B20 in the C - B connective could be traced into the cerebral ganglion, indicating that B20 is a buccal-cerebral interneuron (BCI). Further details of the axonal processes within the cerebral ganglion could not be resolved.

Electrophysiological characteristics In isolated ganglion preparations B20 is generally silent, having a resting potential of - 4 0 to - 5 0 mV. The neuron occasionally receives spontaneous EPSPs but they usually are not of sufficient amplitude to generate an action potential. As detailed below, three types of input were found capable of activating B20: (1) mechanical stretch of the muscles and other tissues of the buccal mass, particularly the pharynx; (2) firing of cerebral-to-buccal interneurons that drive BMPs; and (3) spontaneously occurring rhythmic buccal activity. B20 is incorporated in a BMP elicited by stretch of the pharynx In a reduced preparation, in which the pharynx and the anterior part of the esophagus were left attached to the ganglia (connected via the esophageal nerves and buccal nerves 1), a brief (1-2 s) stretch of the pharyngeal tissue evoked 2 - 3 cycles of patterned activity in B20 (Fig. 4A) and in numerous neurons, including: (1)

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Fig. 4. B20 is incorporated in a BMP elicited by stretching the pharynx. A: in a preparation in which the pharynx and the anterior esophagus were left attached to the buccal ganglion, stretching the pharyngeal musculature elicits two to three cycles of a BMP. The program was monitored by recording from several neurons that are involved in aspects of feeding: lip motor neuron CI1 in the cerebral ganglion; unidentified buccal motor neuron (MN); multiaction neuron B4; and, dopamine-containing neuron B20. Note that the first indications of the program (depolarization and spiking activityin B4) occurred before B20 commenced firing. B: compressing the same region of the pharyngeal tissue did not induce activity in these cells, suggesting that the program is activated selectivelyby stretch receptors in the tissue, and not by mechanical stimulation in general.

buccal neuron B4, a complex, sensory and motor interneuron 12'18'38, (2) an unidentified buccal motor neuron (MN) in the ventral motor neuron cluster; and (3) lip motor neuron C l l in the cerebral ganglion 33. As shown in Fig. 4, the first indications of the buccal program (depolarization of B4) appeared before action potentials occurred in B20, suggesting that B20 did not initiate the program. In addition, functionally eliminating one B20 neuron by injecting hyperpolarizing current did not preclude patterned activity in the other neurons. In contrast to stretching the pharynx, compressing (squeezing) the same region produced only a few PSPs in B20 and in the other neurons, but did not promote patterned activity in any of the buccal or cerebral cells (Fig. 4B). Similarly, tactile (glass rod) or combined chemo-tactile (seaweed) stimulation of the inside of the pharynx did not lead to activity in these neurons (not shown).

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Fig. 3. Camera lucida drawing of an electrophysiologicallyidentified B20 neuron, which had 5(6)-carboxyfluorescein iontophoretically ejected into its soma. Note that B20 is a bipolar neuron with symmetricalprocesses in the cerebral-buccal connectives(C-B conn.) and buccal nerve 3; Note also the numerous branches in the neuropil region of both buccal hemiganglia.

B20 is active during BMPs elicited by stimulation of cerebral command-like neurons In preparations in which the head of the animal remained attached to the ganglia, we found that B20 received excitatory input following tactile or chemical stimulation of the lips and the tentacles. The long latency of the evoked responses suggested that these inputs are indirect, and perhaps are mediated by previously identified cerebral-to-buccal interneurons (CBIs)

231 that drive buccal motor programs 36. Fig. 5 illustrates that B20, indeed, receives synaptic input during activation of CBIs. As shown in the figure, prolonged firing of either CBI-2 (Fig. 5A), or CBI-4 (Fig. 5B), strongly excited B20 and induced rhythmic activity in the pattern characteristic of the evoked BMPs. Note that in both cases activity of B20 occurs early in the programcycle and terminates concurrently with the B4 discharge (activity of B4, however, is not responsible for the fast inhibition that terminates bursting activity of B20). Activity in B20 continued for as long as the cerebral neurons were stimulated and the buccal program persisted. For both CBI-2 and CBI-4, spiking activity of B20 preceded that of B4, but there was a substantial delay between the onset of activity of the cerebral neuron and the occurrence of spikes in B20. Firing CBI-2 or CBI-4 for a short period of time (1-2 s), which in most instances is not sufficient to evoke a BMP, did not evoke activity in B20. Furthermore, in the presence of a solution containing an increased content of calcium ions (6 × Ca 2÷), which increases the amplitude of the evoked postsynaptic potentials, we could not detect short-latency postsynaptic potentials in B20 upon stimulation of either CBI-2 or CBI-4 (not shown). These results suggest that the excitation of B20 is most likely not due to a direct connection from cerebral command-like neurons, but is a result of the activation of the pattern generating network within the buccal ganglion. Stimulation of another cerebral-buccal interneuron, CBI-1, which itself is dopamine-positive and which usually generates a single burst of activity in the buccal ganglion37, also evoked a single burst of activity in B20.

that were tested 5 times each with this stimulus paradigm, 3-4 cycles of a BMP usually occurred when B20 was at resting potential and became activated by CBI-2 activity. In contrast, when B20 was hyperpolarized and thus prevented from becoming activated during the program, we never observed more than one cycle of activity in buccal neurons. These findings indicate that although inactivation of one B20 neuron does not completely abolish the BMP, it diminishes the drive on the pattern generating network and thus attenuates the buccal program. Prolonged depolarization of CBI-2 evokes a sustained BMP that promotes phasic activity in B20 (see Fig. 5A). In order to test for a possible modulatory effect of B20, B20 was fired by depolarizing current (Fig. 6B, thick lines under the records of B20) applied at various phases of the program elicited by CBI-2. The phases of the program are indicated by the horizontal lines, which commence at the inhibitory input in CBI-2 (and high-frequency burst of B4), and terminate at the

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As shown above, B20 is incorporated into the BMP driven by cerebral pattern-initiating neurons CBI-2 and CBI-4. More importantly, B20 has the capacity to modulate these programs. This was demonstrated by stimulating CBI-2 in order to elicit a BMP, and then controlling the firing rate of B20 by injecting either hyperpolarizing or depolarizing current into the neuron. For the first set of experiments, CBI-2 was stimulated with a constant current pulse of 10 sec duration, which usually does not evoke a sustained program. As shown in Fig. 6A1, hyperpolarizing B20 by approximately 30 mV while CBI-2 was stimulated, prevented activation of B20. Under these circumstances only one cycle of a BMP occurred, as evidenced by the single burst in B4. Depolarization of CBI-2 for the same duration while B20 remained at resting potential and thus became strongly activated, lead to 3-4 cycles of rhythmic activity in buccal neurons (Fig. 6A2). In two preparations

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Fig. 5. Cerebral command-like neurons, CBI-2 and CBI-4, elicit rhythmic buccal motor programs that incorporate B20 activity. A: constant current injected into CBI-2 produces rhythmic activity in itself and in buccal neurons B4 and B20. Note that B20 is active in the same p h a s e as CBI-2, whereas B4 is activated antiphasically. B: the BMP evoked by firing of CBI-4 also incorporates B20, as well as B4. Note that the CBI-4 driven program differs in a characteristic manner from that driven by CBI-2.

232 end of the high-frequency burst of B4 (indicated by the horizontal lines at the top of Fig. 6B. Although typically there was some variability of the period of the BMP evoked by CBI-2, out-of-phase activation of B20 appeared to phase advance the program. As shown in Fig. 6B, stimulation of B20 brought in a premature cycle of the BMP (onset indicated by the arrowhead), as evidenced by an early burst of neuron B4 and inhibition in CBI-2. The subsequent bursts were then in phase with the premature burst. Taken together with the findings presented above (Fig. 6A), we conclude that B20 has the capability of modulating the BMP, suggesting that B20 may be part of the buccal pattern generating circuitry for feeding responses. It should be noted, however, that the effects of a single B20 appear to be relatively weak.

fected by firing of B20 include B8, a neuron (or pair of similar neurons) that appears to be involved in radula closing29'35; identified buccal neuron B4; accessory radula closer muscle motor neurons, B15 and B167; and, cerebral lip motor neurons, C l l and C1233. B16 fired in phase with B20 and inspection of individual cycles of the program driven by B20 suggested that some of the fast EPSPs recorded in B16 followed the action potentials in B20 in a one-for-one manner (Fig. 7A). Other neurons, such as B4, discharged antiphasically to the B20 burst (Fig. 7A). In order to test whether B20 is monosynaptically connected to B16, B20 was stimulated while the ganglion was bathed in a solution containing increased concentrations of divalent cations (5 × Ca2+/2 × Mg2+), which raises the threshold of the cells and decreases or blocks polysynaptic transmission. Under these conditions (Fig. 7B), the EPSPs evoked in B16 exhibited synaptic facilitation and followed each action potential in B20 without failures and at unchanged latency, indicative of a monosynaptic connection. B20 also induced EPSPs in motor neuron B8. In one experiment it was shown that

B20 synaptically affects identified buccal motor neurons Brief intracellular stimulation of B20, sufficient to elicit a burst of spikes, provided excitatory input to buccal neuron B4 and to numerous other cells in the buccal and cerebral ganglia. Some of the neurons af-

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Fig. 6. Activity of B20 can modify the CBI-2 driven BMP. A: brief (10 s) stimulation of CBI-2 elicited a single cycle of a BMP activity recorded in neuron B4 when B20 was hyperpolarized (A1). When B20 was kept at resting potental (A 2) stimulation of CBI-2 (same parameters as in A 1) induced 3 cycles of BMP activity recorded in neurons B20 and B4. B: during an ongoing BMP driven by continuous depolarization of CBI-2, B20 was fired at times it would normally be silent (horizontal lines below B20 trace). The B20 stimulation phase advanced the BMP, as evidenced by an early burst in B4 and inhibition in CBI-2 (arrowheads). Beginning with the phase advanced cycle (thick vertical line) caused by stimulation of B20, the BMP continued at regular intervals. The phases of the BMP are indicated by the vertical lines above B20. The lines demark the high frequency discharge of neuron B4 which coincides with inhibition in B20 and CBI-2 (dashed vertical lines). During ingestion, this particular phase of the BMP is thought to correspond to the phase of backward rotation of the radula (Rosen et al., unpublished observations).

233 these EPSPs also persisted in an A S W solution containing a high concentration of divalent cations (Fig. 7C). Activity of B20 evoked synaptic activity in various cerebral cells that are involved in aspects of feeding and which receive synaptic input during BMPs. Such neurons include the lip motor neurons C l l and C12, and several E cluster neurons, many of which are motor neurons for the extrinsic muscles of the buccal mass 5'17. These connections most likely are polysynaptic, since they could not be resolved in the presence of an ASW solution containing high divalent cations.

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B20 is capable of driving a BMP Sustained depolarization of B20 evoked a rhythmic BMP during which B20, as well as numerous other buccal neurons, exhibited cycles of excitation or inhibition (Fig. 8A). The rhythmic program persisted as long as the cell remained depolarized beyond threshold (Fig. 8), and in three experiments activity was maintained for over 5 min of stimulation. In addition to buccal neurons, the program driven by B20 also incorporated cerebral neurons, including command-like neurons (e.g. CBI-2 and CBI-4) that initiate rhythmic buccal motor activity (see Fig. 8B), and lip motor

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Fig. 7. B20 affects identified buccal motor neurons. A: activity of buccal motor neurons B4, B15 and B16 during an ongoing buccal program elicited by depolarization of B20. Note that the excitation of neurons B4 and B15 is antiphasic to that of B20, whereas the initial activity of B16 is in phase with B20. EPSPs in buccal motor neuron B16 appear to occur one for one with the action potentials in B20. B: in a solution containing increased concentrations of divalent cations (5 X Ca 2 + / 2 x Mg 2+ ), spikes in B20 still produced one-for-one facilitating EPSPs in B16, suggesting a monosynaptic connection between B20 and B16. C: u n d e r the same conditions as in B, B20 also produced one-for-one EPSPs in identified motor neuron B8.

Fig. 8. Stimulation of B20 elicits a rhythmic buccal motor program. A: prolonged firing of B20 by means of intracellular injection of depolarizing current, promotes rhythmic activity in various buccal ganglion neurons, such as identified multifunctional neuron B4, and two unidentified buccal neurons in the ventral motor neuron cluster (MN-1 and MN-2). The BMP was sustained for as long as B20 was kept depolarized. B: B20 evokes a typical BMP, monitored by buccal neurons B4 and unidentified motor neuron, MN. The B20 driven program also induced in-phase patterned activity in the cerebral command-like neuron for feeding, CBI-2.

neurons such as C l l . The BMP generated by stimulation of B20 had a period of about 15-20 sec, and similar to BMPs driven by other command-like neurons, the period is moderately variable. The rate of the BMP evoked by B20 is somewhat slower than that of a typical biting program observed in the feeding animal 41. The rate is closer to that of rejection responses, but the interburst intervals are more regular than that observed during rejection 47.

The dopamine antagonist ergonovme blocks the program elicited by B20 The ability of B20 to drive a BMP may be due to the release of its primary transmitter, most likely dopamine. Alternatively, the program may be driven by other transmitters that are co-localized within the cell and

234 A

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Fig. 9. Dopamine-receptor antagonist ergonovine blocks program evoked by stimulation of B20. The typical BMP evoked by stimulation of B20 (A) was blocked during bath application of 10-8 M ergonovine(B). During the block, neither the unidentified motor neuron (MN, top trace) nor identified neuron B4, generated action potentials and rhythmic activityduring B20 stimulation. The capacity of B20 to evoke a BMP was restored when the ergonivinewas washed out with ASW (C).

released, in addition to dopamine. We have obtained pharmacological evidence that is consistent with a role of dopamine in the generation of the program. As shown in Fig. 9, bath application of the dopamine-receptor antagonist, ergonovine 1, blocked the B20 driven program (n = 6). At concentrations of 10 -7 and 10 -8 M, the block produced by ergonovine was at least partially reversible, whereas at higher concentrations (10 -6 M), no reversal was observed following 2 h of washout. Similar blocking effects of ergonovine have been reported in Limax, and long recovery periods for the reversal of the effect were reported 49. Even though depolarization of B20 in the presence of 10 -7 M ergonovine no longer drove a BMP, B20 action potentials still evoked EPSPs in postsynaptic cell, B8. This observation is consistent with the finding that ergonovine selectively blocks the inhibitory effects of dopamine and does not affect excitatory synapses ~.

The BMP driven by B20 is independent of the cerebral ganglion As mentioned before, B20 has two axons that project into the cerebral ganglion, the ganglion that contains neurons that are capable of driving BMPs. Hence, the capacity of B20 to drive a BMP could be due to it activating a group of the cerebral command-like neu-

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,

Fig. 10. BMP driven by B20 is independent of the cerebral ganglion. A: typical BMP driven in the intact cerebral-buccal ganglion preparation. B: following isolation of the buccal ganglion by cutting the cerebral-buccal connectives, the program evoked by stimulation of B20 remained largely unchanged.

rons, although the firing rate of a single command-like neuron (CBI-2) during the BMP driven by B20 does not appear to be sufficiently strong 36 to drive a program (see Fig. 8B). We therefore examined the BMP elicited by B20 before and after the buccal ganglion was surgically isolated from the cerebral ganglion by bilaterally sex,ering both C - B connectives. In Fig. 10A, the typical BMP elicited in an intact cerebral and buccal ganglion preparation by depolarizing B20 is shown. Bursts of phasic activity occurred in B20, along with correlated bursts in identified motor neuron B4, and one unidentified buccal motor neuron (MN). After bilateral 'transsection of the connectives (Fig. 10B), current injection into B20 still drove a similar BMP. Thus, B20 is capable of driving a BMP independent of the cerebral ganglion and cerebral ganglion commandlike neurons. We have not attempted to determine if there are reliable differences in the programs elicited in the isolated buccal ganglion vs. the intact preparation. Our evidence indicates that the effects of dopamine on the cerebral ganglion can be dissociated from the actions of B20 and the possible release of dopamine in the buccal ganglion. DISCUSSION Our results suggest that identified neuron B20 is a pattern generating element associated with the feeding circuitry within the buccal ganglion of Aplysia. B20 is capable of driving a sustained, rhythmic BMP, and is also recruited into BMPs driven by previously identified cerebral command-like neurons implicated in feeding. Several lines of evidence support the idea that B20 is an integral part of a buccal motor pattern generator associated with feeding responses: (1) firing of B20 reliably drives a BMP. (2) B20 is incorporated into a variety of BMPs driven by several means, including: firing of cerebral-to-buccal interneurons, CBI-2 and CBI-4; mechanical stimulation associated with stretch of the pharynx; and, spontaneously occurring

235 buccal programs. (3) B20 can modify the BMP driven by CBI-2. (4) B20 has pre-motor neuron functions, providing synaptic input to various buccal motor neurons. The buccal ganglion is capable of generating at least three different programs associated with feeding: biting, swallowing and rejection 2°, and the results reported here do not permit us determine which one (or more) of these programs B20 is involved in. It is noteworthy however, that the relative timing of excitatory input to B4, B15 and B16 during a program elicited by firing of B20 (Fig. 7), is similar to that typically exhibited by programs elicited by cerebral neuron CBI-2, and there is evidence that the program driven by CBI-2 is associated with biting movements 34. There is no evidence yet that B20 is necessary for these programs to occur, since we have not been able to bilaterally remove B20 from the circuit, and the effects of unilateral hyperpolarization become apparent only under certain circumstances. If cerebral command-like neuron CBI-2 is fired at a rate that evokes only a few cycles of the BMP, and one B20 neuron is removed from the circuitry, the number of cycles of the buccal program is greatly reduced (see Fig. 6A). These results suggest that B20 is an element of a central pattern generating network, or at least modulates some network. Since the effects of manipulating a single neuron under normal firing conditions are rather weak, one can speculate that there is a considerable degree of redundancy within the neuronal circuitry mediating feeding responses. Putative pattern generating neurons for buccal motor programs have been described previously in the buccal ganglion of Aplysia 19,31,40, and other molluscs 2'3'9'13'24-27'50. In Aplysia, one example of buccal neurons that can drive a program is the B31/B32 neurons 4°. In some instances firing of the B31/B32 neurons evoked only one or two cycles, but in other instances the B31/32 cells did evoke a sustained program. Similar to B20, the B31/B32 cells become incorporated in the program that they evoke and are rhythmically active during programs that are driven by stimulation of other pattern initiating neurons 36. The B31/B32 cells, in contrast to B20, do not exhibit full-sized overshooting action potentials. Other identified neurons, such as the B51 cells, are similar to the B31/B32 cells, and in many instances evoke just a few cycles of buccal ganglion activity19'31. The relationship between the various buccal neurons that can drive rhythmic motor programs in Aplysia remains to be determined. Despite apparent differences, there are some interesting similarities between the pattern generating neurons, B20, B31, and B52. During the buccal

program, all three neurons exhibit prominent phases of depolarization, during which spikes are generated. The depolarization is followed by a massive, abruptly occurring hyperpolarization. In the case of B20 and B31/32, the hyperpolarization sets in simultaneously to the onset of the high frequency discharge of buccal neuron B4 (see Figs. 5, 6A, 7, 8), indicating that these neurons may be active during the early phase of the feeding program, which presumably corresponds to the protraction-phase of the radula (Rosen et al., pers. comm.). In Lymnaea, several neurons have been described that are active during the early phase of feeding and are capable of driving a buccal program 2'3. Interestingly, these Nl-type neurons share some common features with pattern generating neurons B20 and B31 in Aplysia. Similar to B31, the Nl-type neurons do not exhibit full-fledged action potentials. Furthermore, like B20, the Nl-type neurons send an axon in the cerebral-buccal connective, and the programs elicited by stimulation of B20 or the Nl-type neurons neurons are somewhat slower than the normal feeding program. The results reported here support other studies that indicated that dopamine plays a role in molluscan feeding behavior 22'28'49. B20 exhibits dopamine histofluorescence and ergonovine, a drug that specifically blocks certain dopamine-receptors abolishes the motor program driven by B20. It is unclear, however, how ergonovine produces its effects, since the evidence indicates that it primarily or exclusively affects inhibitory dopaminergic connections. It is not unlikely that the feeding cpg requires the action of inhbitiory interneurons, perhaps by using inhibitory rebound. Of course, we can not rule out the possibility that ergonovine is acting at some excitatory synapses, or is producing some non-specific alterations of excitability. We have observed however that, the acetylcholine receptor antagonist hexamethonium, which blocks cholinergic excitatory synapses, does not affect the BMP elicited by stimulation of B20, even when applied at very high concentrations (unpublished observations). Our results indicate that bath application of dopamine to the buccal ganglion fails to drive a BMP. Nevertheless, this observation does not strongly argue against a role of dopamine, since bath application is rather unspecific and the drug may activate extrajunctional receptors that normally are not exposed to dopamine 16'23. Furthermore, B20 fires in a burst pattern, and in order for dopamine to function properly it may need to be presented in a phasic manner to the appropriate cells. Consistent with these speculations are the findings obtained in Lymnaea that indicate that in a cell culture consisting of two interconnected neurons of the respiratory circuit, bath application of

236 dopamine is ineffective in driving a rhythmic program, whereas pulsatile application is effective and can mimic the action of a dopaminergic neuron 42. It is interesting that B20, which projects into the cerebral ganglion, is capable of driving a rhythmic buccal motor program, not unlike that driven by dopamine application to the cerebral ganglion37. The program elicited by application of dopamine to the cerebral ganglion might be due to the activation of dopamine receptors that are normally activated by the release of dopamine from terminals of B20 in the cerebral ganglion. The program elicited by stimulation of neuron B20, however, can occur independently of the cerebral ganglion, and can be evoked in the surgically isolated buccal ganglion. Possible connections of B20 to cells in the cerebral ganglion may thus amplify the program elicited by B20, but these cerebral connections are not necessary. Acknowledgements.

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