Distinct mechanisms produce functionally complementary actions of neuropeptides that are structurally related but derived from different precursors.

Many bioactive neuropeptides containing RFamide at their C terminus have been described in both invertebrates and vertebrates. To obtain insight into the functional logic of RFamide signaling, we investigate it here in the feeding system of Aplysia. We focus on the expression, localization, and actions of two families of RFamide peptides, the FRFamides and FMRFamide, in the central neuronal circuitry and the peripheral musculature that generate the feeding movements. We describe the cloning of the FRFamide precursor protein and show that the FRFamides and FMRFamide are derived from different precursors. We map the expression of the FRFamide and FMRFamide precursors in the feeding circuitry using in situ hybridization and immunostaining and confirm proteolytic processing of the FRFamide precursor by mass spectrometry. We show that the two precursors are expressed in different populations of sensory neurons in the feeding system. In a representative feeding muscle, we demonstrate the presence of both FRFamides and FMRFamide and their release, probably from the processes of the sensory neurons in the muscle. Both centrally and in the periphery, the FRFamides and FMRFamide act in distinct ways, apparently through distinct mechanisms, and nevertheless, from an overall functional perspective, their actions are complementary. Together, the FRFamides and FMRFamide convert feeding motor programs from ingestive to egestive and depress feeding muscle contractions. We conclude that these structurally related peptides, although derived from different precursors, expressed in different neurons, and acting through different mechanisms, remain related to each other in the functional roles that they play in the system.

Cycle-to-cycle variability of neuromuscular activity in Aplysia feeding behavior.

Aplysia consummatory feeding behavior, a rhythmic cycling of biting, swallowing, and rejection movements, is often said to be stereotyped. Yet closer examination shows that cycles of the behavior are very variable. Here we have quantified and analyzed the variability at several complementary levels in the neuromuscular system. In reduced preparations, we recorded the motor programs produced by the central pattern generator, firing of the motor neurons B15 and B16, and contractions of the accessory radula closer (ARC) muscle while repetitive programs were elicited by stimulation of the esophageal nerve. In other similar experiments, we recorded firing of motor neuron B48 and contractions of the radula opener muscle. In intact animals, we implanted electrodes to record nerve or ARC muscle activity while the animals swallowed controlled strips of seaweed or fed freely. In all cases, we found large variability in all parameters examined. Some of this variability reflected systematic, slow, history-dependent changes in the character of the central motor programs. Even when these trends were factored out, however, by focusing only on the differences between successive cycles, considerable variability remained. This variability was apparently random. Nevertheless, it too was the product of central history dependency because regularizing merely the high-level timing of the programs also regularized many of the downstream neuromuscular parameters. Central motor program variability thus appears directly in the behavior. With regard to the production of functional behavior in any one cycle, the large variability may indicate broad tolerances in the operation of the neuromuscular system. Alternatively, some cycles of the behavior may be dysfunctional. Overall, the variability may be part of an optimal strategy of trial, error, and stabilization that the CNS adopts in an uncertain environment.

Neuromuscular modulation in Aplysia. I. Dynamic model.

Many physiological systems are regulated by complex networks of modulatory actions. Here we use mathematical modeling and complementary experiments to study the dynamic behavior of such a network in the accessory radula closer (ARC) neuromuscular system of Aplysia. The ARC muscle participates in several types of rhythmic consummatory feeding behavior. The muscle's motor neurons release acetylcholine to produce basal contractions, but also modulatory peptide cotransmitters that, through multiple cellular effects, shape the contractions to meet behavioral demands. We construct a dynamic model of the modulatory network and examine its operation as the motor neurons fire in realistic patterns that change gradually over an hour-long meal and abruptly with switches between the different feeding behaviors. The modulatory effects have very disparate dynamical time scales. Some react to the motor neuron firing only over many cycles of the behavior, but one key effect is fast enough to respond to each individual cycle. Switches between the behaviors are therefore followed by rapid relaxations along some modulatory dimensions but not others. The trajectory of the modulatory state is a transient throughout the meal, ranging widely over regions of the modulatory space not accessible in the steady state. There is a pronounced history-dependency: the modulatory state associated with a cycle of a particular behavior depends on when that cycle occurs and what behaviors preceded it. On average, nevertheless, each behavior is associated with a different modulatory state. In the following companion study, we add a model of the neuromuscular transform to reconstruct and evaluate the actual modulated contraction shapes.

Neuromuscular modulation in Aplysia. II. Modulation of the neuromuscular transform in behavior.

In this work we use mathematical modeling and complementary experiments to study the dynamics of modulation in the accessory radula closer (ARC) neuromuscular system of Aplysia. Here we join a dynamic model of the modulation from the preceding paper to a model of the basal neuromuscular transform (NMT). The resulting complete model of the NMT allows us to predict, test, and analyze the actual modulated contraction shapes in different types of feeding behavior, through entire quasi-realistic meals. The model reproduces a variety of published and new experimental observations. We find that components of the modulatory network act in interdependency and mutual complementarity, one or another playing a key role depending on the behavior and its past history. The history is remembered by slow dynamical components whose persistence prepares the system for future behavior of the same kind. The persistence becomes counterproductive, however, when the behavior suddenly changes. Superposition of fast dynamical components alleviates the problem under most, but not all, circumstances. In the quasi-realistic meals, the modulation improves functional performance on average, but degrades it after certain behavioral switches, when the model predicts sharp contraction transients. These are indeed seen in the real muscle. We propose that the real system does not switch the underlying motor neuron firing patterns abruptly, but relaxes them gradually, matching the relaxation of the peripheral modulatory state, through such behavioral transitions. We model food-induced arousal, a known phenomenon of this kind. The peripheral dynamics of the modulated NMT thus constrain the motor commands of the CNS.

Multiple presynaptic and postsynaptic sites of inhibitory modulation by myomodulin at ARC neuromuscular junctions of Aplysia.

The functional activity of even simple cellular ensembles is often controlled by surprisingly complex networks of neuromodulators. One such network has been extensively studied in the accessory radula closer (ARC) neuromuscular system of Aplysia. The ARC muscle is innervated by two motor neurons, B15 and B16, which release modulatory peptide cotransmitters to shape ACh-mediated contractions of the muscle. Previous analysis has shown that key to the combinatorial ability of B15 and B16 to control multiple parameters of the contraction is an asymmetry in their peptide modulatory actions. B16, but not B15, releases myomodulin, which, among other actions, inhibits the contraction. Work in single ARC muscle fibers has identified a distinctive myomodulin-activated K current as a candidate postsynaptic mechanism of the inhibition. However, definitive evidence for this mechanism has been lacking. Here, working with the single fibers and then motor neuron-elicited excitatory junction potentials (EJPs) and contractions of the intact ARC muscle, we have confirmed two central predictions of the K-current hypothesis: the myomodulin inhibition of contraction is associated with a correspondingly large inhibition of the underlying depolarization, and the inhibition of both contraction and depolarization is blocked by 4-aminopyridine (4-AP), a potent and selective blocker of the myomodulin-activated K current. However, in the intact muscle, the experiments revealed a second, 4-AP-resistant component of myomodulin inhibition of both B15- and B16-elicited EJPs. This component resembles, and mutually occludes with, inhibition of the EJPs by another peptide modulator released from both B15 and B16, buccalin, which acts by a presynaptic mechanism, inhibition of ACh release from the motor neuron terminals. Direct measurements of peptide release showed that myomodulin also inhibits buccalin release from B15 terminals. At the level of contractions, nevertheless, the postsynaptic K-current mechanism is responsible for much of the myomodulin inhibition of peak contraction amplitude. The presynaptic mechanism, which is most evident during the initial build-up of the EJP waveform, underlies instead an increase of contraction latency.