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PubMed2005

Temperature compensation of neuromuscular modulation in aplysia.

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.

Animals↗
PubMed2002

Cerebral CBM1 neuron contributes to synaptic modulation appearing during rejection of seaweed in Aplysia kurodai.

The Japanese species Aplysia kurodai feeds well on Ulva but rejects Gelidium with distinctive rhythmic patterned movements of the jaws and radula. We have previously shown that the patterned jaw movements during the rejection of Gelidium might be caused by long-lasting suppression of the monosynaptic transmission from the multiaction MA neurons to the jaw-closing (JC) motor neurons in the buccal ganglia and that the modulation might be directly produced by some cerebral neurons. In the present paper, we have identified a pair of catecholaminergic neurons (CBM1) in bilateral cerebral M clusters. The CBM1, probably equivalent to CBI-1 in A. californica, simultaneously produced monosynaptic excitatory postsynaptic potentials (EPSPs) in the MA and JC neurons. Firing of the CBM1 reduced the size of the inhibitory postsynaptic currents (IPSCs) in the JC neuron, evoked by the MA spikes, for >100 s. Moreover, the application of dopamine mimicked the CBM1 modulatory effects and pretreatment with a D1 antagonist, SCH23390, blocked the modulatory effects induced by dopamine. It could also largely block the modulatory effects induced by the CBM1 firing. These results suggest that the CBM1 may directly modulate the synaptic transmission by releasing dopamine. Moreover, we explored the CBM1 spike activity induced by taste stimulation of the animal lips with seaweed extracts by the use of calcium imaging. The calcium-sensitive dye, Calcium Green-1, was iontophoretically loaded into a cell body of the CBM1 using a microelectrode. Application of either Ulva or Gelidium extract to the lips increased the fluorescence intensity, but the Gelidium extract always induced a larger change in fluorescence compared with the Ulva extract, although the solution used induced the maximum spike responses of the CBM1 for each of the seaweed extracts. When the firing frequency of the CBM1 activity after taste stimulation was estimated, the Gelidium extract induced a spike activity of ~30 spikes/s while the Ulva extract induced an activity of ~20 spikes/s, consistent with the effective firing frequency (>25 spikes/s) for the synaptic modulation. These results suggest that the CBM1 may be one of the cerebral neurons contributing to the modulation of the basic feeding circuits for rejection induced by the taste of seaweeds such as Gelidium.

Animals↗
PubMed2003

Frequency-dependent regulation of afferent transmission in the feeding circuitry of Aplysia.

During rhythmic behaviors, sensori-motor transmission is often regulated so that there are phasic changes in afferent input to follower neurons. We study this type of regulation in the feeding circuit of Aplysia. We characterize effects of the B4/5 interneurons on transmission from the mechanoafferent B21 to the radula closer motor neuron B8. In quiescent preparations, B4/5-induced postsynaptic potentials (PSPs) can block spike propagation in the lateral process of B21 and inhibit afferent transmission. B4/5 are, however, active during the retraction phase of motor programs, i.e., when mechanoafferent transmission to B8 presumably occurs. To determine whether mechanoafferent transmission is necessarily inhibited when B4/5 are active, we characterize the B4/5 firing frequency during retraction and show that, for the most part, it is low (below 15 Hz). There is, therefore, a low probability that spike propagation will be inhibited. The relative ineffectiveness of low frequency activity is not simply a consequence of insufficient PSP magnitude, because a single PSP can block spike propagation. Instead, it is related to the fact that PSPs have a short duration. When B4/5 fire at a low frequency, there is therefore a low probability that afferent transmission in the lateral process of B21 can be inhibited. In conclusion, we demonstrate that afferent transmission will not always be affected when a neuron that exerts inhibitory effects is active. Although a cell may be ineffective when it fires at a low frequency, ineffectiveness is not necessarily a consequence of spike frequency per se. Instead it may be due to spike timing.

Animals↗
PubMed2003

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.

Animals↗
PubMed2003

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.

Animals↗
PubMed1980

Physiological basis of feeding behavior in Tritonia diomedea. II. Neuronal mechanisms.

1. The nudibranch mollusk Tritonia diomedea feeds by biting and drawing pieces of coelenterate sea whips into its buccal cavity. It then swallows them in a series of cyclic ingestion movements. The coordinated pattern of swallowing can be elicited in reduced preparations by stimulation of identifiable neurons in cerebral or buccal ganglia. 2. Swallowing normally proceeds as a cyclic pattern of protraction and retraction movements driven by alternating bursts in odontophore-radula protractor and retractor motor neurons (MNs), with simultaneous coordination of many other major neurons. 3. Brief or prolonged constant-current stimulation of certain individual buccal ganglion (SW) premotor neurons reliably triggers one or more complete cycles of impulse bursts in motor neurons with coordinated movements of the jaws and buccal mass. The MN bursts produced in this way resemble the ones seen during feeding. Some SW neurons trigger responses that greatly exceed the duration of the stimulus; others drive cyclic output in the motor system whose duration corresponds closely to the duration of the depolarizing current. As well, the rate of MN bursting can be modulated by steady currents of different amplitudes injected into these SW neurons. There are, however, alternative sensory and motor routes independent of SW neurons by which the motor pattern may be elicited and modulated. 4. A search for the source of the feeding rhythm resulted in the identification of a pair of motor neurons (B5) that have prominent burst and pattern-generating capabilities independent of SW activation. They fire vigorous impulse bursts on rebound from spontaneously occurring IPSP waves or from imposed hyperpolarizing potentials of similar amplitude and duration. 5. Constant prolonged depolarizing currents injected into B5 result in the production of repetitive bursts in other MNs that resemble in amplitude and time course those seen during feeding. Cyclic bursting generated in this way apparently entrains other parts of the feeding motor generator since a coordinated output of appropriately phased hyperpolarizing waves, antagonistic EPSP volleys, motor bursts, and movements is simultaneously recorded in other, unstimulated motor neurons of the system. However, the SW neurons are not always entrained by such MN-generated bursting. 6. Brief stimulation of B5 (sufficient to elicit one or a few impulses) also results in the appearance of a depolarizing afterpotential, which is itself capable of regenerating further impulses. When prolonged, constant currents are applied, the impulses that result form bursts whose durations and interburst intervals depend on the amplitude of the stimulating currents. The spike and burst-generating qualities of these neurons (which may include endogenous and interactive components) are sufficient to serve as a source of timing and phasing for the motor pattern.

Animals↗
PubMed1982

Control of feeding motor output by paracerebral neurons in brain of Pleurobranchaea californica.

1. A population of interneurons that control feeding behavior in the mollusk Pleurobranchaea has been analyzed by dye injection and intracellular stimulation/recording in whole animals and reduced preparations. The population consists of 12-16 somata distributed in two bilaterally symmetrical groups on the anterior edge of the cerebropleural ganglion (brain). On the basis of their position adjacent to the cerebral lobes, these cells have been named paracerebral neurons (PCNs). This study concerns pme subset pf [MCs. the large, phasic ones, which have the strongest effect on the feeding rhythm (21). 2. Each PCN sends a descending axon via the ipsilateral cerebrobuccal connective to the buccal ganglion. Axon branches have not been detected in other brain or buccal nerves and hence the PCNs appear to be interneurons. 3. In whole-animal preparations, tonic intracellular depolarization of the PNCs causes them to discharge cyclic bursts of action potentials interrupted by a characteristic hyperpolarization. In all specimens that exhibit feeding behavior, the interburst hyperpolarization is invariably accompanied by radula closure and the beginning of proboscis retraction (the "bite"). No other behavorial effect of PCN stimulation has been observed. 4. In whole-animal preparations, the PCNs are excited by food and tactile stimulation of the oral veil, rhinophores, and tentacles. When such stimuli induce feeding the PCNs discharge in the same bursting pattern seen during tonic PCN depolarization, with the cyclic interburst hyperpolarization phase locked to the bit. When specimens egest an unpalatable object by cyclic buccal movements, however, the PCNs are silent. The PCNs therefore exhibit properties expected of behaviorally specific "command" neurons for feeding. 5. Silencing one or two PCNs by hyperpolarization may weaken but does not prevent feeding induced by natural food stimuli. Single PCNs therefore can be sufficient but are not necessary to induction of feeding behavior. Instead the PCNs presumably operate as a population to control feeding. 6. In isolated nervous system preparations tonic extracellular stimulation of the stomatogastric nerve of the buccal ganglion elicits a cyclic motor rhythm that is similar in general features to the PNC-induced motor rhythm. Bursts of PCN action potentials intercalated at the normal phase position in this cycle intensify the buccal rhythm. Bursts of PCN impulses intercalated at abnormal phase positions reset the buccal rhythm. The PCNs, therefore, also exhibit properties expected of pattern-generator elements and/or coordinating neurons for the buccal rhythm. 7. The PCNs are recruited into activity when the buccal motor rhythm is elicited by stomatogastric nerve stimulation or stimulation of the reidentifiable ventral white cell. The functional synergy between the PCNs and the buccal rhythm is therefore reciprocal. 8...

Animals↗
PubMed1984

Motor organization in pharynx of Helix pomatia.

Identified motor neurons in the buccal ganglia of Helix pomatia and pharynx muscles innervated by them were studied with intracellular recording and cobalt staining. Retrograde cobalt staining via the buccal nerves indicated that neurons occupy relatively constant positions within the ganglia. With intracellular cobalt staining it was shown that the shape of a representative motor neuron (B4) is similar in different preparations. In some cases, however, deviations from the normal pattern of axon distribution were found. Presumed motor endings of neuron B4 in the muscle were also visualized with intracellular staining. Recordings from individual motor neurons show typical phase relationships of spontaneous spike activity. Most motor neurons are active in the retraction phase of the radula. Only excitatory motor neurons were found. Most neurons directly supply more than one muscle. Amplitude of excitatory junction potentials (EJP) and plasticity at neuromuscular junctions from one neuron are similar in different muscles. Single muscle fibers receive polyneuronal innervation. Activity of single motor neurons already leads to muscle contraction even without spiking of the muscle cells. Muscle tension depends on integrated EJP size. Most motor neurons supply typical combinations of a set of muscles. Thus, several muscles can be activated synchronously by activity of a single motor neuron. In this way muscle combinations are predetermined morphologically by the peripheral branching patterns of the respective neurons.

Animals↗
PubMed1993

Pharmacologically induced elements of the hunting and feeding behavior in the pteropod mollusk Clione limacina. II. Effects of physostigmine.

1. A contact of the pteropod mollusk Clione limacina with its prey (small pteropod mollusk Limacina helicina) evokes a complex pattern of hunting and feeding behavior: protraction of tentacles to seize the prey, activation of buccal apparatus to swallow the prey, activation of locomotor system (speeding up of wing beating), reversal of reaction to tactile stimulation of the head, loss of normal (vertical) orientation in space, and swimming in circles. After injection of physostigmine (PhS), the acetylcholinesterase inhibitor, into the hemocoel of intact Clione, all these manifestations of the hunting and feeding behavior could be evoked by tactile stimulation of the head, or they arose spontaneously. 2. In the preparation of the isolated CNS, the effect of PhS on the neural networks controlling different aspects of the hunting and feeding behavior was studied by recording from neurons monitoring activity of different networks (< or = 4 neurons simultaneously). Tactile stimulation of the head was mimicked by a short-term electrical stimulation of the corresponding nerve. Before PhS application, the nerve stimulation evoked elements of the defense reaction, i.e., long-lasting inhibition of all main motor control systems: the locomotor network in the pedal ganglia, the tentacle control network in the cerebral ganglia, and the network controlling radula and hook movements in the buccal ganglia. However, after PhS application, the same stimulus evoked a long-lasting bout of excitation in all the three networks accompanied by activation of the heart-exciting neuron as well as by a modification of the activity of statocyst receptor cells controlling Clione's spatial orientation (the "fictive hunting bout"). Similar hunting bouts could arise spontaneously. 3. Injection of acetylcholine (ACh) into the hemocoel of intact Clione was less effective than injection of PhS. After ACh injection, reversal of reaction to head stimulation was observed in < or = 20% of the experiments (the percentage of positive results was higher if ACh was injected into the head just over the CNS). Bath application of ACh to the isolated CNS did not produce the hunting bouts. However, a short-term local application of ACh to the cerebral ganglia in the isolated CNS resulted in activation of the main motor systems controlling locomotion, protraction of tentacles, and movements of buccal mass. 4. During spontaneous PhS-induced bouts, excitation of different networks involved in hunting behavior was sometimes not quite synchronous. Different networks could be excited in variable order over a period of up to several seconds.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed1994

Modulatory role for the serotonergic cerebral giant cells in the feeding system of the snail, Lymnaea. I. Fine wire recording in the intact animal and pharmacology.

1. The role of the paired serotonergic cerebral giant cells (CGCs) in the feeding system of Lymnaea was examined by electrophysiological and pharmacological techniques. 2. The firing characteristics of the CGCs were recorded by fine wires attached to their cell bodies in freely moving intact snails (in vivo recording) and their "physiological" rates of firing determined during feeding and other behaviors. 3. The mean CGC firing rates recorded in vivo varied between 1 and 20 spikes/min but never reached the average rates seen in the isolated CNS (60-120 spikes/min). Maximum rates of firing were seen during bouts of radula biting/rasping movements characteristic of the consummatory phase of feeding (15 +/- 1.69 spikes/min, mean +/- SE, range 7-20 spikes/min), with lower rates seen during locomotion (6.7 +/- 0.75 spikes/min; range 5-9 spikes/min. The cells were rarely active when the animal was quiescent (1.45 +/- 0.91 spikes/min; range 0-2 spikes/min). 4. In vivo recorded CGC firing was phase locked to the feeding movements of the animal, with spikes occurring just before the opening of the mouth, during the protraction phase of the feeding cycle. 5. Evoking firing rates on the CGCs in the isolated preparation similar to those seen in vivo during rasping movements (7-20 spikes/min) did not elicit a fictive feeding pattern in an inactive preparation. Neither did bath application of 10(-9) M serotonin (5-HT; the transmitter of the CGCs). 6. To allow the modulatory role of the CGCs to be examined during patterned activity, the fictive feeding pattern was evoked in the isolated preparation by injecting depolarizing current into a modulatory neuron, the slow oscillator (SO). 7. The tonic firing activity of the CGCs was accurately maintained by current injection in the isolated preparation at rates equivalent to that occurring during feeding, locomotion, and quiescence in the intact snail. This was possible where the CGCs became silent after 1-2 h. Only when the CGCs activity was maintained at a rate (approximately 15 spikes/min) similar to that occurring during rasping, was the SO able to drive a full, high-frequency fictive feeding pattern (15-20 cycles/min). At lower rates of CGC firing, the SO-driven rhythm was either of lower frequency or no rhythm occurred at all (CGCs silent). 8. In many isolated preparations (80%) the CGCs remained active, and it was difficult to maintain specific levels of tonic activity by current injection.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Neuromuscular regeneration by buccal motoneuron B15 after peripheral nerve crush in Aplysia californica.

1. We studied regeneration of neuromuscular connections by identified buccal motoneuron B15 after axotomy produced by crushing nerve 4; the intact contralateral nerve 4 served as control. Electrophysiological recordings, intracellular dye injections, and light and electron microscopy were used to characterize the nature and time course of neuromuscular reinnervation as well as the fate of the isolated distal stump of the motor axon. 2. Axonal outgrowth or sprouting in the form of numerous "regenerites" occurred from the proximal stump of the transected B15 axon, and these regenerites projected through the crush site along the length of the nerve to innervate target muscles at the periphery. 3. Reinnervation of one of the target muscles, the accessory radula closer (I5), was first detected 3 wk after nerve crush. Neuromuscular excitatory postsynaptic potentials measured in individual I5 muscle fibers were initially small and approached control amplitudes by 8 wk postlesion. Newly regenerated neuromuscular synapses displayed facilitation and depression to repeated B15 stimulation with properties similar to those of control synapses, even at early times postlesion. 4. Reinnervation of other buccal muscles by B15, such as I4, appeared slightly delayed relative to that observed for I5. No evidence of abnormal or enlarged fields of innervation were observed, and as in control preparations, regenerated neuromuscular connections were strictly limited to muscles ipsilateral to the B15 cell body. 5. Physiological evidence suggested that the distal axon stumps of B15, although isolated from their cell bodies, survive for several weeks after axotomy. In addition, several large axon profiles indicative of motor axons were seen in cross-sections of nerve 4 taken close to the muscle and distal to the crush site, indicating survival of distal axon stumps. 6. When B15 was selectively stimulated, the newly formed regenerites failed to fire the distal axon stump of B15, demonstrating that the regenerites do not reinnervate the distal stump. 7. Degeneration of axons in nerve 4 distal to the crush site was observed in cross-sections of the nerve at 8 wk postlesion; using ultrathin sections we found cellular debris in individual axon profiles as well as large acellular masses within nerve 4, the latter likely representing the concretion of many axons. Additional evidence for such degenerative changes appeared in the form of autofluorescing spherical bodies or "spheroids" both in individual axons and the nerve distal to the crush site.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

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.

Acetylcholine↗
PubMed1995

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.

Animals↗
PubMed1995

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)

Amino Acid Sequence↗
PubMed1996

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.

Acetylcholine↗
PubMed1996

B64, a newly identified central pattern generator element producing a phase switch from protraction to retraction in buccal motor programs of Aplysia californica.

1. Buccal motor programs in Aplysia are characterized by two phases of activity, which represent protraction and retraction of the radula in intact animals. The shift from protraction to retraction is caused by synaptic activity inhibiting neurons that are active during protraction and exciting neurons that are active during retraction. 2. B64, a newly identified neuron present bilaterally in the buccal ganglia, is partially responsible for the phase shift. Stimulating a single B64 causes bilateral inhibition of neurons B31/B32 and other neurons active during protraction and cause bilateral excitation of neurons B4/B5 and other neurons active during retraction. B64 is active throughout retraction. The amplitude and waveforms of the synaptic potentials caused by firing B64 are similar, but not identical, to those seen during retraction. 3. Some of the effects of B64 on B31/B32 and on B4/B5 are monosynaptic, as shown by their maintained presence in high divalent cation seawater, which blocks polysynaptic activity. 4. A brief depolarization of B64 leads to a long-lasting depolarization and firing. The ability of B64 to respond in this way is at least partially caused by an endogenous plateau potential, as this property is still seen after synaptic transmission is blocked. 5. Hyperpolarization of B64 bilaterally and preventing the somata from firing unmasks a large excitatory postsynaptic potential in B64. This procedure does not block the shift from protraction to retraction, indicating that spiking in the B64 somata is not necessary for the phase shift. 6. The firing pattern and synaptic connections of B64 are consistent with the hypothesis that the neuron is part of a central pattern generator underlying buccal motor programs. B64 is monosynaptically inhibited by neurons that are active along with B31/B32, which are responsible for producing the protraction phase of a buccal motor program. During the later portion of the protraction phase B64 is excited. In addition, firing B64 can phase advance and phase delay buccal motor programs. 7. Regulating the firing of B64 can regulate the expression of buccal motor programs. Stimulation of B64 at frequencies of 0.5-1.0 Hz leads to complete inhibition of buccal motor programs, whereas steady-state depolarization of B64 can lead to repetitive bursts of activity.

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PubMed1999

Actions of a pair of identified cerebral-buccal interneurons (CBI-8/9) in Aplysia that contain the peptide myomodulin.

A combination of biocytin back-fills of the cerebral-buccal connectives and immunocytochemistry of the cerebral ganglion demonstrated that of the 13 bilateral pairs of cerebral-buccal interneurons in the cerebral ganglion, a subpopulation of 3 are immunopositive for the peptide myomodulin. The present paper describes the properties of two of these cells, which we have termed CBI-8 and CBI-9. CBI-8 and CBI-9 were found to be dye coupled and electrically coupled. The cells have virtually identical properties, and consequently we consider them to be "twin" pairs and refer to them as CBI-8/9. CBI-8/9 were identified by electrophysiological criteria and then labeled with dye. Labeled cells were found to be immunopositive for myomodulin, and, using high pressure liquid chromatography, the cells were shown to contain authentic myomodulin. CBI-8/9 were found to receive synaptic input after mechanical stimulation of the tentacles. They also received excitatory input from C-PR, a neuron involved in neck lengthening, and received a slow inhibitory input from CC5, a cell involved in neck shortening, suggesting that CBI-8/9 may be active during forward movements of the head or buccal mass. Firing of CBI-8 or CBI-9 resulted in the activation of a relatively small number of buccal neurons as evidenced by extracellular recordings from buccal nerves. Firing also produced local movements of the buccal mass, in particular a strong contraction of the I7 muscle, which mediates radula opening. CBI-8/9 were found to produce a slow depolarization and rhythmic activity of B48, the motor neuron for the I7 muscle. The data provide continuing evidence that the small population of cerebral buccal interneurons is composed of neurons that are highly diverse in their functional roles. CBI-8/9 may function as a type of premotor neuron, or perhaps as a peptidergic modulatory neuron, the functions of which are dependent on the coactivity of other neurons.

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PubMed1999

Electrophysiological and behavioral analysis of lip touch as a component of the food stimulus in the snail Lymnaea.

Electrophysiological and video recording methods were used to investigate the function of lip touch in feeding ingestion behavior of the pond snail Lymnaea stagnalis. Although this stimulus was used successfully as a conditioning stimulus (CS) in appetitive learning experiments, the detailed role of lip touch as a component of the sensory stimulus provided by food in unconditioned feeding behavior was never ascertained. Synaptic responses to lip touch in identified feeding motoneurons, central pattern generator interneurons, and modulatory interneurons were recorded by intracellular electrodes in a semi-intact preparation. We showed that touch evoked a complex but characteristic sequence of synaptic inputs on each neuron type. Touch never simply activated feeding cycles but provided different types of synaptic input, determined by the feeding phase in which the neuron was normally active in the rhythmic feeding cycle. The tactile stimulus evoked mainly inhibitory synaptic inputs in protraction-phase neurons and excitation in rasp-phase neurons. Swallow-phase neurons were also excited after some delay, suggesting that touch first reinforces the rasp then swallow phase. Video analysis of freely feeding animals demonstrated that during normal ingestion of a solid food flake the food is drawn across the lips throughout the rasp phase and swallow phase and therefore provides a tactile stimulus during both these retraction phases of the feeding cycle. The tactile component of the food stimulus is strongest during the rasp phase when the lips are actively pressed onto the substrate that is being moved across them by the radula. By using a semi-intact preparation we demonstrated that application of touch to the lips during the rasp phase of a sucrose-driven fictive feeding rhythm increases both the regularity and frequency of rasp-phase motoneuron firing compared with sucrose applied alone.

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