Accessory Radula Closer Muscle Research Articles

Identification of a New Neuropeptide Precursor Reveals a Novel Source of Extrinsic Modulation in the Feeding System ofAplysia

The Aplysia feeding system is advantageous for investigating the role of neuropeptides in behavioral plasticity. One family of Aplysia neuropeptides is the myomodulins (MMs), originally purified from one of the feeding muscles, the accessory radula closer (ARC). However, two MMs, MMc and MMe, are not encoded on the only known MM gene. Here, we identify MM gene 2 (MMG2), which encodes MMc and MMe and four new neuropeptides. We use matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to verify that these novel MMG2-derived peptides (MMG2-DPs), as well as MMc and MMe, are synthesized from the precursor. Using antibodies against the MMG2-DPs, we demonstrate that neuronal processes that stain for MMG2-DPs are found in the buccal ganglion, which contains the feeding network, and in the buccal musculature including the ARC muscle. Surprisingly, however, no immunostaining is observed in buccal neurons including the ARC motoneurons. In situ hybridization reveals only few MMG2-expressing neurons that are mostly located in the pedal ganglion. Using immunohistochemical and electrophysiological techniques, we demonstrate that some of these pedal neurons project to the buccal ganglion and are the likely source of the MMG2-DP innervation of the feeding network and musculature. We show that the MMG2-DPs are bioactive both centrally and peripherally: they bias egestive feeding programs toward ingestive ones, and they modulate ARC muscle contractions. The multiple actions of the MMG2-DPs suggest that these peptides play a broad role in behavioral plasticity and that the pedal-buccal projection neurons that express them are a novel source of extrinsic modulation of the feeding system of Aplysia.

Temperature Compensation of Neuromuscular Modulation inAplysia

Physiological systems that must operate over a range of temperatures often incorporate temperature-compensatory mechanisms to maintain their output within a relatively narrow, functional range of values. We analyze here an example in the accessory radula closer (ARC) neuromuscular system, a representative part of the feeding neuromusculature of the sea slug Aplysia. The ARC muscle's two motor neurons, B15 and B16, release, in addition to ACh that contracts the muscle, modulatory peptide cotransmitters that, through a complex network of effects in the muscle, shape the ACh-induced contractions. It is believed that this modulation is critical in optimizing the performance of the muscle for successful, efficient feeding behavior. However, previous work has shown that the release of the modulatory peptides from the motor neurons decreases dramatically with increasing temperature. From 15 to 25 degrees C, for example, release decreases 20-fold. Yet Aplysia live and feed successfully not only at 15 degrees C, but at 25 degrees C and probably at higher temperatures. Here, working with reduced B15/B16-ARC preparations in vitro as well as a mathematical model of the system, we have found a resolution of this apparent paradox. Although modulator release decreases 20-fold when the temperature is raised from 15 to 25 degrees C, the observed modulation of contraction shape does not decrease at all. Two mechanisms are responsible. First, further downstream within the modulatory network, the modulatory effects themselves-experimentally dissected by exogenous modulator application-have temperature dependencies opposite to that of modulator release, increasing with temperature. Second, the saturating curvature of the dose-response relations within the network diminishes the downstream impact of the decrease of modulator release. Thus two quite distinct mechanisms, one depending on the characteristics of the individual components of the network and the other emerging from the network's structure, combine to compensate for temperature changes to maintain the output of this physiological system.

Tight or Loose Coupling Between Components of the Feeding Neuromusculature ofAplysia?

Like other complex behaviors, the cyclical, rhythmic consummatory feeding behaviors of Aplysia-biting, swallowing, and rejection of unsuitable food-are produced by a complex neuromuscular system: the animal's buccal mass, with numerous pairs of antagonistic muscles, controlled by the firing of numerous motor neurons, all driven by the motor programs of a central pattern generator (CPG) in the buccal ganglia. In such a complex neuromuscular system, it has always been assumed that the activities of the various components must necessarily be tightly coupled and coordinated if successful functional behavior is to be produced. However, we have recently found that the CPG generates extremely variable motor programs from one cycle to the next, and so very variable motor neuron firing patterns and contractions of individual muscles. Here we show that this variability extends even to higher-level parameters of the operation of the neuromuscular system such as the coordination between entire antagonistic subsystems within the buccal neuromusculature. In motor programs elicited by stimulation of the esophageal nerve, we have studied the relationship between the contractions of the accessory radula closer (ARC) muscle, and the firing patterns of its motor neurons B15 and B16, with those of its antagonist, the radula opener (I7) muscle, and its motor neuron B48. There are two separate B15/B16-ARC subsystems, one on each side of the animal, and these are indeed very tightly coupled. Tight coupling can, therefore, be achieved in this neuromuscular system where required. Yet there is essentially no coupling at all between the contractions of the ARC muscles and those of the antagonistic radula opener muscle. We interpret this result in terms of a hypothesis that ascribes a higher-order benefit to such loose coupling in the neuromusculature. The variability, emerging in the successive feeding movements made by the animal, diversifies the range of movements and thereby implements a trial-and-error search through the space of movements that might be successful, an optimal strategy for the animal in an unknown, rapidly changing feeding environment.

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.

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.

Ion currents and mechanisms of modulation in the radula opener muscles of Aplysia.

Ion currents and mechanisms of modulation in the radula opener muscles of Aplysia. J. Neurophysiol. 78: 2372-2387, 1997. Numerous studies of plasticity in the feeding behavior of Aplysia have shown that substantial plasticity is due to peripheral neuromodulation of the feeding musculature. Extensive previous work focusing on the accessory radula closer (ARC) muscle has led to the realization that a major function of the modulation in that muscle may be to ensure efficient coordination between its contractions and those of its antagonist muscles. For a more complete understanding, therefore, we must study these muscles also. Here we have studied the radula opener muscles I7-I10. Using single isolated muscle fibers under voltage clamp, we have characterized ion currents gated by voltage and by the physiological contraction-inducing neurotransmitter acetylcholine (ACh) and the effects of the physiological modulators serotonin, myomodulins A and B, and FMRFamide. Our results explain significant aspects of the electrophysiological behavior of the whole opener muscles, as well as why the opener and ARC muscles behave similarly in many ways yet differently in some key respects. Opener muscles express four types of K currents: inward rectifier, A-type [IK(A)], delayed rectifier [IK(V)], and Ca2+-activated [IK(Ca)]. They also express an L-type Ca current [ICa] and a leakage current. ACh activates a positive-reversing cationic current [IACh(cat)] and a negative-reversing Cl current [IACh(Cl)]. The opener muscles differ from the ARC in that, in the openers, activation of IK(A) occurs approximately 9 mV more positive and there is much less IACh(Cl). In both muscles, IACh(cat) most likely serves to depolarize the muscle until ICa activates to supply Ca2+ for contraction, but further depolarization and spiking is opposed by coactivation of IK(A), IK(V), IK(Ca), and IACh(Cl). Thus the differences in IK(A) and IACh(Cl) may well be key factors that prevent spikes in the ARC but often allow them in the opener muscles. As in the ARC, the modulators enhance ICa and so potentiate contractions. They also activate a modulator-specific K current, which causes hyperpolarization and depression of contractions. Finally, in the opener muscles but not in the ARC, the modulators activate a depolarizing cationic current that may help phase-advance the contractions. Each modulator exerts these effects to different degrees and thus has a distinct effect on voltage and contraction size and shape. The overall effect then will depend on the specific combinations of modulators released in different behaviors. By understanding the modulation in the opener muscles, as well as in the ARC, we are now in a position to understand how the behavior of the two muscles is coordinated under a variety of circumstances.

Two ion currents activated by acetylcholine in the ARC muscle of Aplysia.

1. This work continues our examination of the electrophysiology and contractions of single fibers dissociated from a widely studied molluscan muscle, the accessory radula closer (ARC) muscle of Aplysia californica, aimed at understanding its excitation-contraction mechanisms and their modulation. 2. Extensive previous work has characterized a number of basal ion currents present in the fibers and effects of transmitters and peptide cotransmitters that modulate ARC-muscle contractions in vivo. Here we use current clamp, voltage clamp, and contraction measurements to examine the actions of acetylcholine (ACh), the transmitter that induces the contractions. 3. As in the whole ARC muscle, ACh depolarizes unclamped fibers maximally to about -25 mV where, no matter how much ACh is applied, the depolarization saturates. 4. The underlying ACh-activated current is in fact the sum of two quite distinct components, IACh,cat and IACh,Cl. 5. IACh,cat is itself a mixed current carried by cations (physiologically mainly by Na+, but to a significant degree also by Ca2+), reverses near +20 mV, rectifies inwardly, exhibits prominent voltage-dependent kinetics of activation with hyperpolarization, and is selectively blocked by hexamethonium. 6. In contrast, IACh,Cl is carried by Cl-, reverses near -60 mV, exhibits little rectification or voltage-dependent kinetics, is activated selectively by suberyldicholine, and is blocked by alpha-bungarotoxin. 7. Both currents activate fast when ACh is applied, desensitize relatively slowly in its presence, then deactivate fast. Both currents are activated at similar ACh concentrations, half-maximally at approximately 10 microM. Both currents also are activated by carbachol and propionylcholine and blocked by d-tubocurarine, bicuculline and paraoxon. Picrotoxin and atropine block IACh,cat better, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and anthracene 9-carboxylic acid IACh,Cl better. 8. The two currents are virtually identical to ACh-activated cationic (Na) and Cl currents that are ubiquitous in molluscan neurons. As has been proposed for the neuronal currents, IACh,cat resembles vertebrate neuronal nicotinic ACh-receptor (nAChR) currents, whereas IACh,Cl resembles vertebrate skeletal muscle nAChR currents. 9. Functionally, we believe that IACh,cat serves primarily to depolarize the ARC muscle to open voltage-activated L-type Ca channels, allow Ca2+ influx, and initiate contraction. Physiologically significant Ca2+ may also enter through the ACh,cat channels themselves. 10. By superimposing on IACh,cat, IACh,Cl brings the reversal potential of the combined current to around -25 mV and thereby sets a relatively negative upper limit to the ACh-induced depolarization. We propose that this is its physiological role. By limiting the depolarization, IACh,Cl limits the degree of activation of the Ca current and Ca2+ influx, and so prevents excessive contraction. More importantly, it moderates the voltage during contraction to a range where small voltage changes can finely grade contraction amplitude in this nonspiking muscle. 11. Indeed, in contraction experiments on the single fibers, there is an inverse correlation between the IACh,Cl/IACh,cat ratio and the magnitude of the ACh-induced depolarization and contraction. Furthermore, increased pharmacological activation of IACh,Cl depresses, and block of IACh,Cl enhances, both the depolarization and contraction. 12. Obligatory simultaneous coactivation of IACh,cat and IACh,Cl in the ARC muscle may be part of a peripheral control mechanism that automatically keeps the size of its contractions within behaviorally optimal limits.

Nine members of the myomodulin family of peptide cotransmitters at the B16-ARC neuromuscular junction of Aplysia.

1. Neuromodulation by multiple related peptides with different spectra of physiological effects appears an effective way to integrate complex physiological functions. A good opportunity to examine this issue occurs in the accessory radula closer (ARC) neuromuscular circuit of Aplysia, where, extensive previous work has shown, acetylcholine-induced contractions of the muscle are variously modulated by several families of peptide cotransmitters released under appropriate behavioral circumstances from the muscle's own two motor neurons. 2. In this work we focused on the myomodulins (MMs) released from motor neuron B16. Previous work has characterized MMA (PMSMLRLamide) and MMB (GSYRMMRLamide). We now similarly purified from ARC neuromuscular material and sequenced MMC (GWSMLRLamide), MMD (GLSMLRLamide), MME (GLQMLRLamide), and MMF (SLNMLRLamide). Three additional MMs, MMG (TLSMLRLamide), MMH (GLHMLRLamide), and MMI (SLSMLRLamide), are encoded by a known MM gene. B16 probably synthesizes, and coreleases, all nine MMs. Further MMs have been found in other mollusks. All evidence indicates that the MMs are a major, widely distributed family of molluscan neuropeptides important as neuromuscular modulators and probably also central transmitters or modulators. 3. MM effects on motor neuron B16-elicited ARC muscle contractions were best analyzed as the sum of three distinct actions: potentiation, depression of the amplitude of the contractions, and acceleration of their relaxation rate. We compared the effectiveness of all nine MMs in these respects. We correlated this with their effectiveness in enhancing the L-type Ca current and activating a specific K current in voltage-clamped dissociated ARC muscle fibers, effects we previously proposed to underlie, respectively, the potentiation and the depression of contractions. 4. All nine MMs were similarly effective in enhancing the Ca current and, as far as it was possible to determine, potentiating the amplitude as well as accelerating the relaxation rate of the contractions. 5. In contrast, the MMs' ability to activate the K current and depress the contractions varied greatly. MMB and MMC, in particular, were weak, whereas the other seven MMs were considerably more effective in both respects. 6. Altogether, we were able to explain the potentiating and depressing strengths of the MMs by the magnitude of their modulation of the Ca and K currents, providing further support for our hypothesis that the effects on contraction amplitude are mediated by the effects on the two currents. 7. The net effect on contraction amplitude was determined by the balance between the potentiation and depression. Although most MM concentrations had both potentiating and depressing actions, potentiated contractions predominated at low and depressed contractions (but with accelerated relaxation rate) at high concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)

Ca(2+)-activated K current in the ARC muscle of Aplysia.

1. This work continues our examination of the electrophysiology and contractions of single, functionally intact fibers dissociated from a widely studied molluscan muscle, the accessory radula closer (ARC) muscle of Aplysia californica, aimed at understanding its excitation-contraction mechanisms and their modulation. Extensive previous work has characterized a number of ion currents in the fibers. 2. Here we describe an additional major current that could not be studied earlier because, unlike all of the other currents in the ARC muscle fibers, it becomes prominent only during contraction of the fiber. It is a Ca(2+)-activated K current, associated with contraction most likely because both are activated by the same elevation in intracellular free Ca2+. 3. We used several manipulations to elicit the Ca(2+)-activated K current and contraction: depolarizing voltage steps in the presence of extracellular Ca2+, application of caffeine in the presence or absence of extracellular Ca2+ (and thus presumably working by releasing Ca2+ from intracellular stores), application of the Ca(2+)-ionophore A23187, and direct iontophoretic injection of Ca2+ into the fiber. 4. The Ca(2+)-activated K current reversed around -70 mV, not far from EK, and the reversal potential shifted substantially with elevated extracellular K+. Activation of the current was not only Ca2+ dependent, but also quite strongly voltage dependent, promoted by depolarization. The current was well blocked by tetraethylammonium (KD approximately 2 mM), but not blocked by even 10 mM 4-aminopyridine or low concentrations of the K-current blocking toxins charybdotoxin and apamin. 5. After a depolarizing voltage step in Ca(2+)-containing solution, the Ca(2+)-activated K current appeared, often with some delay, as a large peak of current that soon disintegrated into a prolonged period of individual oscillatory transients of Ca(2+)-activated K current, sometimes correlated with transient contractions. Similar transients could be elicited by caffeine or iontophoretic Ca2+ injection. More extensive study of the underlying Ca2+ dynamics will be presented elsewhere, but we interpret these phenomena in terms of our hypothesis that the ARC muscle generates both contraction and the Ca(2+)-activated K current by Ca(2+)-induced Ca2+ release (CICR), in which a small depolarization-induced influx of extracellular Ca2+ releases more Ca2+ from intracellular stores. 6. The Ca(2+)-activated K current is significant in the physiological operating voltage range of the ARC muscle, and its predicted hyperpolarizing action and consequent negative-feedback depression of contractions is likely to be an important part of the integrated set of mechanisms that regulate the muscle's contractility.

FRF peptides in the ARC neuromuscular system of Aplysia: purification and physiological actions.

1. One preparation that has proven to be advantageous for the study of neuromuscular modulation is the accessory radula closer (ARC) muscle of Aplysia californica and its motor neurons B15 and B16. In this study three members of a new peptide family have been purified from this well-characterized preparation. Because these peptides terminate in Phe-Arg-Phe-amide, we have named them FRFA, FRFB, and FRFC. The FRFs are thus RFamide peptides and are related to the widely studied neuropeptide FMRFamide. 2. The FRFs are present in the ARC motor neuron B15 in small quantities. 3. When they are exogenously applied, the FRFs decrease the size of ARC muscle contractions elicited by stimulation of either motor neuron B15 or B16. They appear to do this by a combination of presynaptic and postsynaptic actions. 4. Presynaptically, the FRFs appear to act like the buccalins, another family of inhibitory ARC neuropeptides. Both families of peptides reduce the size of motor neuron-elicited excitatory junction potentials (EJPs) presumably by decreasing presynaptic acetylcholine (ACh) release. 5. Postsynaptically, the FRFs appear to depress contractions because they activate a characteristic voltage-dependent, 4-amino-pyridine-sensitive K current in the ARC muscle. The same current is activated by a second class of ARC modulators: those that exert potentiating actions at low doses and inhibitory actions at high doses, i.e., serotonin, the small cardioactive peptides (SCPs), and particularly the myomodulins. Receptors mediating activation of the K current by the FRFs and the other modulators do, however, appear to be different. 6. We hypothesize that the inhibitory actions of the FRFs prevent excessively large muscle contractions. If contraction size is limited, then contraction duration is also limited. This may allow faster and more energetically favorable switching between contractions of antagonistic muscles.

Myomodulin application increases cAMP and activates cAMP-dependent protein kinase in the accessory radual closer muscle of Aplysia

Myomodulin A (MM A) application or stimulation of neuron B16, which releases MM A, increases cAMP levels in the accessory radula closer (ARC) muscle of Aplysia. MM A application also increases cAMP-dependent protein kinase (cAPK) activity in one subcellular compartment of the muscle. These results suggest that at least part of MM A's effects in this system are mediated via the cAPK signal transduction pathway. Since the effects of the small cardioactive peptides (SCPs) on ARC muscle contraction are similar to those of MM A, our results suggest that the convergent physiological effects of MM A and SCP B in this system may be due, in part, to the two peptide neuromodulators utilizing the same signal transduction pathway.

Enhancement of Ca current in the accessory radula closer muscle of Aplysia californica by neuromodulators that potentiate its contractions

A major goal of neuroscience is to identify the neural and cellular mechanisms of behavior and its plasticity. Progress toward this goal has come particularly from work with a small number of tractable model preparations. One of these is the simple neuromuscular circuit consisting of the accessory radula closer (ARC) muscle of the mollusk Aplysia californica and its innervating motor and modulatory neurons. Contraction of the ARC muscle underlies a component of Aplysia feeding behavior, and plasticity of the behavior is in large part due to modulation of the amplitude and duration of the contractions of the muscle by a variety of modulatory neurotransmitters and peptide cotransmitters, among them the small cardioactive peptides (SCPs), myomodulins (MMs), and serotonin (5-HT). We have studied single dissociated ARC muscle fibers in order to determine whether modulation of membrane ion currents in the muscle might underlie these effects. First, we confirmed that the dissociated fibers were functionally intact: just as with the whole ARC muscle, their contractions were potentiated by 5-HT and SCPB and potentiated as well as depressed by MMA, and their cAMP content was greatly elevated by 5-HT, SCPA and SCPB, and to a lesser extent by MMA and MMB. Next, using voltage-clamp techniques, we found that two ion currents present in the fibers were indeed modulated. The fibers possess a dihydropyridine-sensitive, high-threshold "L"-type Ca current. This current was enhanced by the modulators that potentiate ARC-muscle contractions--5-HT, SCPA and SCPB, and MMA and MMB--but not by buccalinA, a modulator that does not act directly on the ARC muscle. All of the potentiating modulators, as well as elevation of cAMP in the fibers by forskolin or a cAMP analog, maximally enhanced the current about twofold and mutually occluded each other's effects. Since the Ca current supplies Ca2+ necessary for contraction of the muscle, the enhancement of the current is a good candidate to be a major mechanism of the potentiation of the contractions. In the following article we report that the modulators also, to different degrees, activate a distinctive K current and thereby depress the contractions. Net potentiation or depression then depends on the balance between the relative strengths of the modulation of the two ion currents.

Activation of K current in the accessory radula closer muscle of Aplysia californica by neuromodulators that depress its contractions

The neural and cellular mechanisms of plasticity apparent in the feeding behavior of the mollusk Aplysia californica have been extensively studied in a simple neuromuscular circuit consisting of the accessory radula closer (ARC) muscle and its innervating motor and modulatory neurons. In this circuit, the plasticity is largely due to modulation of the amplitude and duration of the contractions of the muscle by a variety of modulatory neurotransmitters and peptide cotransmitters, among them the small cardioactive peptides (SCPs), myomodulins (MMs), and serotonin (5-HT). We have studied dissociated but functionally intact ARC muscle fibers to determine whether modulation of membrane ion currents in the muscle might underlie these effects. Using voltage-clamp techniques, we found that two currents were indeed modulated. In the preceding article, we proposed that enhancement of "L"-type Ca current is the mechanism by which the modulators potentiate the amplitude of ARC-muscle contractions. Here, we report that the modulators also activate a distinctive K current. Large K currents were activated, in particular, by MMA, while MMB, the SCPs, and 5-HT activated much smaller currents most likely of the same kind. Buccalins, modulators that do not act directly on the ARC muscle, were ineffective. The modulator-induced K current was strongly enhanced by depolarization, but relatively slowly so that its amplitude continued to increase for several hundred milliseconds following a depolarizing voltage step. The current was Ca2+ independent, not readily blocked by extracellular Cs+ or Ba2+ and only by high concentrations of tetraethylammonium. However, it was almost completely blocked by as little as 10 microM 4-aminopyridine. In contrast to the modulator-induced enhancement of Ca current, activation of the K current was not significantly mimicked by elevation of cAMP. In the intact as well as the dissociated ARC muscle, although low levels of all of the modulators potentiate contractions, even moderate levels of MMA strongly depress them, whereas the other modulators depress them weakly only at high concentrations. The modulator-induced K current appears well suited to counteract depolarization of the muscle and thus limit activation of the "L"-type Ca current that provides Ca2+ essential for contraction. We therefore propose that the modulators depress ARC-muscle contractions in large part by activating the K current. This occurs simultaneously with the enhancement of the Ca current; net potentiation or depression then depends on the balance between the relative strengths of the modulation of the two ion currents.

Characterization of the membrane ion currents of a model molluscan muscle, the accessory radula closer muscle of Aplysia california. I. Hyperpolarization-activated currents.

1. The simple neuromuscular circuit consisting of the accessory radula closer (ARC) muscle of the mollus Aplysia californica together with its innervating motor and modulatory neurons has been extensively studied as a model preparation in which it might be possible to reach an integrated understanding of the neural and cellular mechanisms of behavioral plasticity, in this case of a component of Aplysia feeding behavior. Previous work has suggested that much of the plasticity of this behavior is implemented by appropriate release of modulatory neurotransmitters and peptide cotransmitters that modulate several parameters of the contractions of the ARC muscle. However, little is as yet known about the underlying cellular mechanisms. 2. We have begun to study single, functionally intact fibers dissociated from the ARC muscle to assess to what extent the modulation of its contraction might be mediated by one candidate mechanism, modulation of its membrane ion currents. First, however, it was necessary to gain a thorough understanding of the unmodulated currents and their likely roles in normal contraction. Using voltage-clamp techniques, we have therefore identified and characterized the major currents present in the ARC muscle fibers. We describe these currents in this and the following two papers. These results constitute the first detailed description of ion currents in a molluscan muscle and lay the foundation for further study, to be presented in subsequent papers, of the roles of two currents that we have indeed found to be modulated in ways likely to contribute to the modulation of contraction. 3. In this paper we first describe the general electrophysiological characteristics of the dissociated fibers and present evidence that the fibers can be adequately space clamped. 4. The physiological operating voltage range of the nonspiking ARC muscle most likely extends from about -80 to about -25 mV. The steady-state current-voltage (I-V) relation of total membrane current rectifies inwardly in the negative and outwardly in the positive portion of this voltage range, with a plateau region of high or even negative slope resistance separating the two regions of rectification. 5. The current responsible for the inward rectification at negative voltages is a classical inwardly rectifying K current. It is activated by hyperpolarization with quasi-instantaneous kinetics; its whole I-V relation shifts along the voltage axis in a Nernstian manner with altered extracellular K+ concentration; it is blocked by low extracellular Ba2+ and Cs+, and the block is promoted by hyperpolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of the membrane ion currents of a model molluscan muscle, the accessory radula closer muscle of Aplysia californica. III. Depolarization-activated Ca current.

1. The accessory radula closer (ARC) muscle of Aplysia californica and its innervation is a model preparation for the study of the neural and cellular mechanisms of behavioral plasticity. Much of the plasticity is due to modulation of contractions of the muscle by a variety of neurotransmitters and peptide cotransmitters. Preliminary to investigating the cellular mechanisms of this modulation, we have characterized the major membrane ion currents present in the unmodulated ARC muscle and their likely roles in normal contraction. We have studied single dissociated but functionally intact ARC muscle fibers under voltage clamp. This is the last of three papers describing this work. In the first paper we characterized two currents prominent at hyperpolarized voltages, a classical inwardly rectifying K current and a Cl current induced by elevated intracellular Cl-. In the second paper we examined two large outward K currents activated at more depolarized voltages, an "A" current and a delayed rectifier. 2. In this paper, we describe an inward depolarization-activated Ca current that underlies and is normally completely masked by the K currents and is revealed when they are blocked. 3. The Ca current begins to activate above -40 or -30 mV. It is fully available for activation at voltages more negative than -60 mV. It activates in milliseconds, then inactivates relatively slowly with maintained depolarization. The current is larger and inactivates slower when it is carried by Ba2+ rather than Ca2+. The inactivation is current rather than voltage dependent. The current is blocked by Co2+, Cd2+, and with a characteristic time dependence, by the dihydropyridine Ca-channel antagonist nifedipine. 4. These properties of the current characterize it as a high-threshold, L-type Ca current. 5. This current most likely provides Ca2+ necessary for contraction of the ARC muscle.

Characterization of the membrane ion currents of a model molluscan muscle, the accessory radula closer muscle of Aplysia californica. II. Depolarization-activated K currents

1. The accessory radula closer (ARC) muscle of Aplysia californica and its innervation is a model preparation for the study of the neural and cellular mechanisms of behavioral plasticity. Much of the plasticity is mediated by release of neurotransmitters and peptide cotransmitters that modulate contractions of the muscle. Preliminary to investigating the cellular mechanisms of action of these modulators, we have characterized the major membrane ion currents present in the unmodulated ARC muscle and their likely roles in normal contraction. We have studied single dissociated but functionally intact ARC muscle fibers under voltage clamp. This is the second of three papers describing this work. In the preceding paper we described the electrophysiological properties of the fibers at hyperpolarized voltages, and characterized the two major hyperpolarized-activated currents present, a classical inwardly rectifying K current and a Cl current induced by elevated intracellular Cl-. 2. In this paper we dissect the large outward current that becomes activated when the fibers are depolarized above -50 or -40 mV. We find that this current consists of two major depolarization-activated K currents, a fast transient "A"-type current and a slower maintained delayed rectifier, with perhaps a small component of Ca(2+)-activated K current. 3. The A current begins to activate with voltage steps above -50 or -40 mV. It activates in milliseconds, then inactivates virtually completely within 100-200 ms. It is fully available for activation below -80 mV, and almost completely inactivated above -40 mV. It is Ca2+ independent, half-maximally blocked by approximately 3 mM 4-aminopyridine (4-AP) but only 460 mM tetraethylammonium (TEA). 4. The delayed rectifier both activates and inactivates more slowly and more positive than the A current. Thus it begins to activate only above -30 or -20 mV; it activates in tens of milliseconds, then inactivates incompletely over several seconds; it is fully available below -70 mV and inactivated above 0 mV. It is Ca2+ independent, half-maximally blocked by 10 mM TEA and 3-10 mM 4-AP. 5. In the following paper we describe a depolarization-activated Ca current that underlies the K currents and most likely provides Ca2+ necessary for contraction of the muscle. By activating simultaneously with the Ca current, the K currents serve to prevent spikes, so that the depolarization is confined to a range where small voltage changes provide fine control over a wide range of contraction strengths.

Buccalin is present in the cholinergic motor neuron B16 of Aplysia and it depresses accessory radula closer muscle contractions evoked by stimulation of B16

The accessory radula closer (ARC) buccal muscle motor neuron B16 is buccalin-immunoreactive and it synthesizes a peptide with chromatographic properties identical to those of buccalin. Terminal varicosities in the ARC muscle are buccalin-immunoreactive, and buccalin reduces muscle contractions evoked by stimulation of neuron B16, probably by a presynaptic action.

Structure and action of buccalin: a modulatory neuropeptide localized to an identified small cardioactive peptide-containing cholinergic motor neuron of Aplysia californica.

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 of biting 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.

Myomodulin: a bioactive neuropeptide present in an identified cholinergic buccal motor neuron of Aplysia.

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 of a 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 peptide--termed 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.