Changes in Aplysia biting responses during food arousal are partially mediated by the serotonergic metacerebral cells (MCCs) The nervous system of the marine mollusc Aplysia provides an advantageous model system for the study of the neural basis ofbehavior and the modification ofbehavior by learning and motivational states such as arousal. We have been studying a form of arousal that is elicited by food and is characterized by general changes in locomotion (1) and cardiovascular responses (2) as well as by specific alterations in feeding behavior-such as progressive increases in the strength and speed of biting (1,3,4).Previous studies (5-7) have demonstrated that changes in biting during food arousal are partially mediated by the serotonergic metacerebral cells (MCCs). The MCCs exert central actions within the nervous system and peripheral actions on buccal muscles. We have studied the peripheral actions of the MCCs on the accessory radula closer muscle (ARCM), a muscle used in biting, which is innervated by the MCC and two cholinergic buccal motor neurons B15 and B16 (8). The MCC is not a motor neuron; its activity does not produce ARCM contractions. Instead it increases the strength of contractions produced by stimulation of neurons B15 and B16, presumably by increasing cAMP levels in the muscle (9).This modulatory input from the MCCs, however, accounts for only a part of the manifestations of arousal, because animals in which the MCCs have been lesioned still exhibit a progressive buildup ofthe magnitude and frequency ofbiting, although to a lesser degree (7). This suggests that arousal may be produced by more than one modulatory system. In fact, we have demonstrated that nerve fibers and varicosities in the ARCM contain the neuropeptide SCPB and that exogenous application of SCPB exerts modulatory actions similar to those of serotonin and the MCC-i.e., SCPB increases cAMP in the ARCM and enhances contractions produced by motor neuron stimulation without itself producing a contraction (10). Therefore, there may be peptidergic as well as serotonergic modulation of the ARCM. As a first step in understanding the functional significance of this dual modulation, we have now identified sources of peptidergic input to the ARCM. METHODS Extraction of Peptides from Motor Neurons. Identified neurons B15 and B16 (8) were marked by iontophoresis of fast green dye. In some experiments, peptides were radiolabeled in vivo (11). Buccal ganglia were incubated with 0.5 mCi of [35S]methionine (1 Ci = 37 GBq; Amersham) for 24 hr at 18°C in 1 ml of 50% artificial sea water (ASW)/50% sterile (0.2-,um filtered) hemolymph/100 ,l of antibiotics (penicillin and streptomycin each at 50 units per ml)/colchicine (2.5 ,ul of 1 M colchicine dissolved in Me2SO). Colchicine was added to inhibit axonal transport, which raised intrasomatic peptide levels and reduced, or eliminated, labeled peptides that might be present in fibers and terminals near the somata of dissected neurons (12). After 24 hr, ganglia were rinsed and incubated i...

Growing evidence suggests that different forms of complex motor acts are constructed through flexible combinations of a small number of modules in interneuronal networks. It remains to be established, however, whether a module simply controls groups of muscles and functions as a computational unit for use in multiple behaviors (behavior independent) or whether a module controls multiple salient features that define one behavior and is used primarily for that behavior (behavior specific). We used the Aplysia feeding motor network to examine the two proposals by studying the functions of identifiable interneurons. We identified three types of motor programs that resemble three types of behaviors that Aplysia produce: biting, swallowing, and rejection. Two ingestive programs (biting, swallowing) are defined by two movement parameters of the feeding apparatus (the radula): one is the same in both programs (phasing of radula closure motoneurons relative to radula protraction-retraction), whereas the other parameter (protraction duration) is different in the two programs. In each program, these two parameters were specified together by an individual neuron, but the neurons in each were different (B40 for biting, B30 for swallowing). These findings support the existence of behavior-specific modules. Furthermore, neuron B51 was found to mediate a phase that can be flexibly added on to both ingestive and egestive-rejection programs, suggesting that B51 may be a behavior-independent module. The functional interpretation of the role played by these modules is supported by the patterns of synaptic connectivity that they make. Thus, both behavior-specific and behavior-independent modules are used to construct complex behaviors.

1. Several lines of evidence suggest that the I7-I10 muscle group contributes to the radula opening phase of behavior in Aplysia; 1) extracellular stimulation of these muscles in reduced preparations causes the halves of the radula to separate, 2) synaptic activity can be recorded from muscles I7-I10 in intact animals when the radula is opening, and 3) motor neurons innervating I7-I10 are activated out of phase with retractor/closer motor neurons during cycles of buccal activity driven by the cerebral-to-buccal interneuron 2 (CBI-2). 2. All of the opener muscles are innervated by the B48 neurons, a bilaterally symmetrical pair of cholinergic motor neurons. B48 neurons produce excitatory junction potentials (EJPs) in opener muscle fibers that summate to produce muscle contractions. Contraction size is determined by the size of depolarization in muscle fibers and/or by action potentials that are triggered by summation of B48-evoked EJPs. 3. In addition to input from B48 neurons, opener muscles also receive excitatory input from the cholinergic multiaction neurons B4/B5. EJPs evoked by stimulation of neurons B4/B5 are 1/10 the size of B48-evoked EJPs. Consequently, changes in muscle tension produced by B4/B5 activity are relatively small. In contrast to B48 neurons, neurons B4/B5 are likely to be active during the closing/retraction phase of behavior. During cycles of buccal activity driven by neuron CBI-2, neurons B4/B5 fire in phase with closer/retractor motor neurons. Thus opener muscles may develop a modest amount of tension during the closing/retraction phase of behavior as a result of synaptic input from neurons B4/B5. 4. Opener muscles may also develop tension during closing/retraction simply by virtue of the fact that they have been stretched. When isolated opener muscles are lengthened, depolarizations are recorded from individual muscle fibers, and muscle tension increases. With sufficient changes in fiber length, action potentials are elicited. These action potentials produce twitchlike muscle contractions that become rhythmic with maintained stretch. Stretch-activated depolarizations are generally first apparent when muscle length is increased by 1 mm. Length changes of 4-5 mm are generally necessary to elicit twitchlike muscle contractions. Changes of 1-2 mm in muscle length are observed when the opener muscle's antagonist, the accessory radula closer, is activated in reduced preparations. 5. Stretch may also modulate B48-induced contractions of the opener muscles. When muscle length is increased, B48-elicited contractions of the I7 muscle are larger. These increases in contraction amplitude are accompanied by decreases in contraction latency. 6. We conclude that muscles I7-I10 contract vigorously in response to strong excitatory input from neuron B48 and contribute to radula opening. Stretch may potentiate this activity. Thus, if radula closer muscles contract vigorously and pull on the opener muscles, the opener muscles will respond by contracting more vigorously themselves. This may be a mechanism fo...

When Aplysia are initially exposed to food stimuli, their biting responses show progressive increases in speed and strength. The accessory radula closer (ARC) buccal muscles have been used to study this phenomenon, and it has been shown that changes in ARC muscle contraction are partially due to activity ofa serotonergic neuron that modulates this muscle, by both a direct action and an action on two ARC motor neurons (B15 and B16). The motor neurons use acetylcholine as their excitatory transmitter, but they also contain bioactive peptides that can potentiate muscle contractions when they are exogenously applied. Motor neuron B15 contains the structurally related small cardioactive peptides A and B, whereas motor neuron B16 contains a different peptidetermed myomodulin. In the present study we determined the full amino acid sequence of myomodulin. Myomodulin is present in the ARC muscle, and exogenous application of the peptide potentiates ARC muscle contractions in a manner similar to the potentiation by small cardioactive peptides A and B. The structure of myomodulin, however, bears little resemblance to the small cardioactive peptides. Thus it appears that ARC muscle contractions may be regulated by at least three distinct classes of neuromodulators: serotonin, the small cardioactive peptides, and myomodulin.An important issue in behavioral neuroscience is the nature of the modulatory synaptic mechanisms that affect behavior. We have taken advantage of the simplicity of the nervous system of Aplysia to study synaptic modulatory processes and their possible role in motivational states. One such motivational state is arousal, which can be induced by exposing an animal to food. Food arousal in Aplysia is characterized by changes in locomotion, posture, and cardiovascular function (1-3), as well as by progressive increases in the speed and strength of biting as the animal begins to feed (1, 4, 5). Modulation of feeding responses has been studied in the experimentally advantageous muscle the accessory radula closer (ARC), which is innervated by two identified motor neurons, B15 and B16 (6). Contraction of the ARC is modulated in part by activity of a serotonergic neuron, the metacerebral cell (7-9).We have begun to investigate possible additional sources of neuromodulation in the ARC muscle (10, 11). We have shown that both of the ARC motor neurons B15 and B16 are cholinergic (6) but also contain neuropeptides that, when exogenously applied, potentiate muscle contractions elicited by motor neurons. We showed that B15 contains the characterized (12)(13)(14) small cardioactive peptides A and B (SCPA and SCPB) and that B16 contains a peptide that has methionine in positions 2 and 4 of its amino acid sequence and, therefore, is not one of the SCPs. This peptide was preliminarily named myomodulin (11). In this study we obtained the complete primary structure of the B16 peptide myomodulin. METHODS Extraction and Purification. ARC muscle (30 g derived from 1000 Aplysia californica) were heated for 10 min at 100'C...

Afferent transmission can be regulated (or gated) so that responses to peripheral stimuli are adjusted to make them appropriate for the ongoing phase of a motor program. Here, we characterize a gating mechanism that involves regulation of spike propagation in Aplysia mechanoafferent B21. B21 is striking in that afferent transmission to the motor neuron B8 does not occur when B21 is at resting membrane potential. Our data suggest that this results from the fact that spikes are not actively propagated to the lateral process of B21 (the primary contact with B8). When B21 is peripherally activated at its resting potential, electrotonic potentials in the lateral process are on average 11 mV. In contrast, mechanoafferent activity is transmitted to B8 when B21 is centrally depolarized via current injection. Our data suggest that central depolarization relieves propagation failure. Full-size spikes are recorded in the lateral process when B21 is depolarized and then peripherally activated. Moreover, changes in membrane potential in the lateral process affect spike amplitude, even when the somatic membrane potential is virtually unchanged. During motor programs, both the lateral process and the soma of B21 are phasically depolarized via synaptic input. These depolarizations are sufficient to convert subthreshold potentials to full-size spikes in the lateral process. Thus, our data strongly suggest that afferent transmission from B21 to B8 is, at least in part, regulated via synaptic control of spike initiation in the lateral process. Consequences of this control for compartmentalization in B21 are discussed, as are specific consequences for feeding behavior.

A model system that consists of a muscle utilized in biting, the accessory radula closer (ARC), and the two cholinergic motor neurons innervating this muscle, neurons B15 and B16, has been used to study the expression of food-induced arousal in the marine mollusk Aplysia. The ARC muscle receives modulatory input from an extrinsic source, the serotonergic metacerebral cells, which partially accounts for the progressive increase in the strength ofbiting seen in aroused animals. Another source of modulation may arise from the ARC motor neurons themselves, which synthesize neuropeptides that can potentiate ARC contractions. Neuron B15 synthesizes the two homologous peptides, small cardioactive peptides A and B, whereas neuron B16 synthesizes the structurally unrelated peptide myomodulin. Here we report the purification and sequencing of a neuropeptide termed buccalin and show that it is colocalized with the small cardioactive peptides to neuron B15. Buccalin is also bioactive at the ARC neuromuscular junction but, in contrast to the small cardioactive peptides, when exogenously applied, it decreases rather than increases the size of muscle contractions elicited by firing of the motor neurons. Also unlike the small cardioactive peptides, which exert postsynaptic actions, buccalin seems to act only presynaptically. It has no effect on muscle relaxation rate and decreases motor neuron-elicited excitatory junction potentials in the ARC without affecting contractions produced by direct application of acetylcholine to the muscle. Neuron B15, therefore, appears to contain three modulatory neurotransmitters, two of which may act postsynaptically on the muscle to potentiate the action of the primary neurotransmitter acetylcholine and one of which may act presynaptically on nerve terminals to inhibit acetylcholine release.We have been studying a motivational state, food-induced arousal, that is manifested in the consummatory biting response of the marine mollusk Aplysia californica as progressive increases in both the speed and strength of biting (1, 2). Work in a model system (3) that consists of one of the muscles utilized in biting, the accessory radula closer (ARC), and the two cholinergic motor neurons that innervate this muscle, buccal motor neurons B15 and B16, has led us to conclude that arousal in the Aplysia biting response is partially mediated by activity of the serotonergic metacerebral cells (4-6).We have also demonstrated that motor neurons B15 and B16 synthesize neuropeptides that have bioactivity at the ARC muscle (7,8). Neuron B15 contains the two structurally homologous (9-11) peptides, small cardioactive peptides A and B (SCPA and SCPB) (7), whereas neuron B16 contains the unrelated peptide myomodulin (8). When exogenously applied, all three peptides produce increases in the size and the relaxation rate of muscle contractions elicited by motor neuron stimulation (7,8,12). Therefore, we have hypothesized that the ARC neuromuscular system is modulated both extrinsically (by means of the metacerebral ...

To gain insights into the physiological role of cotransmission, we measured peptide release from cell B15, a motorneuron that utilizes ACh as its primary transmitter but also contains putative peptide cotransmitters, the small cardioactive peptides (SCPs) and the buccalins (BUCs). All stimulation parameters used were in the range in which B15 fires in freely moving animals. We stimulated neuron B15 in bursts and systematically varied the interburst interval, the intraburst frequency, and burst duration. Both peptides were preferentially released when B15 was stimulated at higher intra- or interburst frequencies or with longer burst durations. Across stimulation patterns, the amount of peptide released depended on the mean frequency of stimulation and was independent of the specific pattern of stimulation. The parameters of stimulation that produce a larger release of peptides correspond to those that evoke larger contractions. Large and frequent contractions are likely to fuse or summate, thus disrupting the rhythmic behavior mediated by the muscle innervated by motorneuron B15. Because the combined effect of the SCPs and BUCs is to accelerate the relaxation and shorten the duration of muscle contractions, these peptides reduce the probability of the disruptive fusion or summation of muscle contractions. Because these cotransmitters regulate an aspect of muscle contractions that is not controlled by acetylcholine (ACh), the primary transmitter of B15, we suggest that peptides and ACh form parallel but functionally distinct lines of transmission at the neuromuscular junction. Both types of transmission may be necessary to ensure that behavior remains efficient over a wide range of conditions.

Many neurons contain multiple peptide cotransmitters in addition to their classical transmitters. We are using the accessory radula closer neuromuscular system of Aplysia, which participates in feeding in these animals, to define the possible consequences of multiple modulators converging on single targets. How these modulators are released onto their targets is of critical importance in understanding the outcomes of their modulatory actions and their physiological role. Here we provide direct evidence that the partially antagonistic families of modulatory peptides, the myomodulins and buccalins, synthesized by motorneuron B16 are costored and coreleased in fixed ratios. We show that this release is calcium-dependent and independent of muscle contraction. Furthermore, we show that peptide release is initiated at the low end of the physiological range of motorneuron firing frequency and that it increases with increasing motorneuron firing frequency. The coordination of peptide release with the normal operating range of a neuron may be a general phenomenon and suggests that the release of peptide cotransmitters may exhibit similar types of regulation and plasticity as have been observed for classical transmitters. Stimulation paradigms that increase muscle contraction amplitude or frequency also increase peptide release from motor neuron B16. The net effect of the modulatory peptide cotransmitters released from motorneuron B16 would be to increase relaxation rate and therefore allow more frequent and/or larger contractions to occur without increased resistance to antagonist muscles. The end result of this modulation could be to maximize the efficiency of feeding.