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.

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...

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...

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.