A newly identified buccal interneuron initiates and modulates feeding motor programs in aplysia.

Despite considerable progress in characterizing the feeding central pattern generator (CPG) in Aplysia, the full complement of neurons that generate feeding motor programs has not yet been identified. The distribution of neuropeptide-containing neurons in the buccal and cerebral ganglia can be used as a tool to identify additional elements of the feeding circuitry by providing distinctions between otherwise morphologically indistinct neurons. For example, our recent study revealed a unique and potentially interesting unpaired PRQFVamide (PRQFVa)-containing neuron in the buccal ganglion. In this study, we describe the morphological and electrophysiological characterization of this novel neuron, which we designate as B50. We found that activation of B50 is capable of producing organized rhythmic output of the feeding CPG. The motor programs elicited by B50 exhibit some similarities as well as differences to motor programs elicited by the command-like cerebral-to-buccal interneuron CBI-2. In addition to activating the feeding CPG, B50 may act as a program modulator.

The enterins: a novel family of neuropeptides isolated from the enteric nervous system and CNS of Aplysia.

To identify neuropeptides that have a broad spectrum of actions on the feeding system of Aplysia, we searched for bioactive peptides that are present in both the gut and the CNS. We identified a family of structurally related nonapeptides and decapeptides (enterins) that are present in the gut and CNS of Aplysia, and most of which share the HSFVamide sequence at the C terminus. The structure of the enterin precursor deduced from cDNA cloning predicts 35 copies of 20 different enterins. Northern analysis, in situ hybridization, and immunocytochemistry show that the enterins are abundantly present in the CNS and the gut of Aplysia. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry we characterized the enterin-precursor processing, demonstrated that all of the precursor-predicted enterins are present, and determined post-translational modifications of various enterins. Enterin-positive neuronal somata and processes were found in the gut, and enterins inhibited contractions of the gut. In the CNS, the cerebral and buccal ganglia, which control feeding, contained the enterins. Enterin was also present in the nerve that connects these two ganglia. Enterins reduced the firing of interneurons B4/5 during feeding motor programs. Such enterin-induced reduction of firing also occurred when excitability of B4/5 was tested directly. Because reduction of B4/5 activity corresponds to a switch from egestive to ingestive behaviors, enterin may contribute to such program switching. Furthermore, because enterins are present throughout the nervous system, they may also play a regulatory role in nonfeeding behaviors of Aplysia.

Sonometric measurements of motor-neuron-evoked movements of an internal feeding structure (the radula) in Aplysia.

In many systems used to study rhythmic motor programs, the structures that generate behavior are at least partially internal. In these systems, it is often difficult to directly monitor neurally evoked movements. As a consequence, although motor programs are relatively well characterized, it is generally less clear how neural activity is translated into functional movements. This is the case for the feeding system of the mollusk Aplysia. Here we used sonomicrometry to monitor neurally evoked movements of the food-grasping organ in Aplysia, the radula. Movements were evoked by intracellular stimulation of motor neurons that innervate radula muscles that have been extensively studied in reduced preparations. Nevertheless our results indicate that the movements and neural control of the radula are more complex than has been assumed. We demonstrate that motor neurons previously characterized as radula openers (B48) and closers (B8, B15, B16) additionally produce other movements. Moreover, we show that the size of the movement evoked by a motor neuron can depend on the preexisting state of the radula. Specifically, the motor neurons B15 and B16 produce large closing movements when the radula is partially open but produce relatively weak closing movements in a preparation at rest. Thus the efficacy of B15 and B16 as radula closers is context dependent.

Serotonergic and peptidergic modulation of the buccal mass protractor muscle (I2) in aplysia.

Plasticity of Aplysia feeding has largely been measured by noting changes in radula protraction. On the basis of previous work, it has been suggested that peripheral modulation may contribute to behavioral plasticity. However, peripheral plasticity has not been demonstrated in the neuromuscular systems that participate in radula protraction. Therefore in this study we investigated whether contractions of a major radula protraction muscle (I2) are subject to modulation. We demonstrate, first, that an increase in the firing frequency of the cholinergic I2 motoneurons will increase the amplitude of the resulting muscle contraction but will not modulate its relaxation rate. We show, second, that neuronal processes on the I2 muscle are immunoreactive to myomodulin (MM), RFamide, and serotonin (5-HT), but not to small cardioactive peptide (SCP) or buccalin. The I2 motoneurons B31, B32, B61, and B62 are not immunoreactive to RFamide, 5-HT, SCP, or buccalin. However, all four cells are MM immunoreactive and are capable of synthesizing MMa. Third, we show that the bioactivity of the different modulators is somewhat different; while the MMs (i.e., MMa and MMb) and 5-HT increase I2 muscle relaxation rate, and potentiate muscle contraction amplitude, MMa, at high concentrations, depresses muscle contractions. Fourth, our data suggest that cAMP at least partially mediates effects of modulators on contraction amplitude and relaxation rate.

Glutamate is the fast excitatory neurotransmitter of small cardioactive peptide-containing Aplysia radula mechanoafferent neuron B21.

B21 is a radula mechanoafferent neuron in the mollusc Aplysia which likely plays a crucial role in integrating environmental cues into the feeding motor program. To facilitate understanding B21's interactions with its postsynaptic followers, we sought to identify its neurotransmitter. We find that B21 makes a chemical synapse onto the follower neuron B8. Although B21-induced excitatory postsynaptic potentials (EPSPs) in B8 paradoxically diminish in amplitude with B8 hyperpolarization, we show that an inwardly rectifying current is responsible. We conclude that these B21-induced EPSPs are likely glutamatergic as they are blocked by the glutamate antagonist DNQX. Furthermore, B8 exhibits a depolarizing response to exogenous glutamate, which is antagonized by DNQX. Finally, exogenous glutamate occludes B21-evoked EPSPs in B8.

Diverse synaptic connections between peptidergic radula mechanoafferent neurons and neurons in the feeding system of Aplysia.

The buccal ganglion of Aplysia contains a heterogeneous population of peptidergic, radula mechanoafferent (RM) neurons. To investigate their function, two of the larger RM cells (B21, B22) were identified by morphological and electrophysiological criteria. Both are low-threshold, rapidly adapting, mechanoafferent neurons that responded to touch of the radula, the structure that grasps food during ingestive and egestive feeding movements. Sensory responses of the cells consisted of spike bursts at frequencies of 8-35 Hz. Each cell was found to make chemical, electrical, or combined synapses with other sensory neurons, motor neurons and interneurons involved in radula closure and/or protraction-retraction movements of the odontophore. Motor neurons receiving input included the following: B8a/b, B15, and B16, which innervate muscles contributing to radula closing; and B82, a newly identified neuron that innervates the anterodorsal region of the I1/I3 muscles of the buccal mass. B21 and B22 can affect buccal motor programs by way of their connections to interneurons such as B19 and B64. Fast, chemical, excitatory postsynaptic potentials (EPSPs) produced by RM neurons, such as B21, exhibited strong, frequency-dependent facilitation, a form of homosynaptic plasticity. Firing B21 also produced a slow EPSP in B15 that increased the excitability of the cell. Thus a sensory neuron mediating a behavioral response may have modulatory effects. The data suggest multiple functions for RM neurons including 1) triggering of phase transitions in rhythmic motor programs, 2) adjusting the force of radula closure, 3) switching from biting to swallowing or swallowing to rejection, and 4) enhancing food-induced arousal.

Outputs of radula mechanoafferent neurons in Aplysia are modulated by motor neurons, interneurons, and sensory neurons.

The gain of sensory inputs into the nervous system can be modulated so that the nature and intensity of afferent input is variable. Sometimes the variability is a function of other sensory inputs or of the state of motor systems that generate behavior. A form of sensory modulation was investigated in the Aplysia feeding system at the level of a radula mechanoafferent neuron (B21) that provides chemical synaptic input to a group of motor neurons (B8a/b, B15) that control closure and retraction movements of the radula, a food grasping structure. B21 has been shown to receive both excitatory and inhibitory synaptic inputs from a variety of neuron types. The current study investigated the morphological basis of these heterosynaptic inputs, whether the inputs could serve to modulate the chemical synaptic outputs of B21, and whether the neurons producing the heterosynaptic inputs were periodically active during feeding motor programs that might modulate B21 outputs in a phase-specific manner. Four cell types making monosynaptic connections to B21 were found capable of heterosynaptically modulating the chemical synaptic output of B21 to motor neurons B8a and B15. These included the following: 1) other sensory neurons, e.g. , B22; 2) interneurons, e.g., B19; 3) motor neurons, e.g., B82; and 4) multifunction neurons that have sensory, motor, and interneuronal functions, e.g., B4/5. Each cell type was phasically active in one or more feeding motor programs driven by command-like interneurons, including an egestive motor program driven by CBI-1 and an ingestive motor program driven by CBI-2. Moreover, the phase of activity differed for each of the modulator cells. During the motor programs, shifts in B21 membrane potential were related to the activity patterns of some of the modulator cells. Inhibitory chemical synapses mediated the modulation produced by B4/5, whereas excitatory and/or electrical synapses were involved in the other instances. The data indicate that modulation is due to block of action potential invasion into synaptic release regions or to alterations of transmitter release as a function of the presynaptic membrane potential. The results indicate that just as the motor system of Aplysia can be modulated by intrinsic mechanisms that can enhance its efficiency, the properties of primary sensory cells can be modified by diverse inputs from mediating circuitry. Such modulation could serve to optimize sensory cells for the different roles they might play.

A proprioceptive role for an exteroceptive mechanoafferent neuron in Aplysia.

Afferent regulation of centrally generated activity is likely to be more complex than has been established. We show that a neuron that is an exteroceptor can also function as a proprioceptor. We study the Aplysia neuron B21. Previous data suggest that B21 functions as an exteroceptor during the radula closing/retraction phase of ingestive feeding. We show that the tissue innervated by B21, the subradula tissue (SRT), is innervated by a motor neuron (B66) and that B66-induced SRT contractions trigger centripetal spikes in B21. Thus, B21 is also a proprioceptor. To determine whether exteroceptive and proprioceptive activities occur during the same phase of ingestive feeding, we further characterize B66. We show that B66 stimulation does not close or retract the radula. Instead it opens it. Moreover, B66 is electrically coupled to other opening/protraction neurons. Finally, we elicit motor programs in semi-intact preparations and show that during radula opening/protraction we observe B66 activity, SRT contractions, and spikes in B21 that can be eliminated if B66 is indirectly hyperpolarized. B21 is, therefore, likely to act as an exteroceptor during one phase of ingestive feeding and as a proprioceptor during the antagonistic phase. Previous experiments have shown that centripetal spikes in B21 are only transmitted to one follower if they are "gated in" by depolarization. During ingestive programs B21 is centrally depolarized during closing/retraction, but it is not depolarized during opening/protraction. We sought to determine whether there are other followers that receive B21 input when it is not centrally depolarized. We found one such cell. Moreover, we found that stimulation of B21 during radula opening/protraction significantly decreases the duration of this phase of behavior. Thus, proprioceptive activity in B21 is likely to have an impact on motor programs.

Peptide cotransmitter release from motorneuron B16 in aplysia californica: costorage, corelease, and functional implications.

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

Modulation of radula opener muscles in Aplysia.

We observed fibers immunoreactive (IR) to serotonin (5-HT), the myomodulins (MMs), and FMRFamide on the I7-I10 complex in the marine mollusk Aplysia californica. The I7-I10 muscle complex, which produces radula opening, is innervated primarily by one motor neuron, B48. B48 is MM-IR and synthesizes authentic MM(A). When B48 is stimulated in a physiological manner, cAMP levels are increased in opener muscles. cAMP increases also are seen when the MMs are applied to opener muscles but are not seen with application of the B48 primary neurotransmitter acetylcholine (ACh). Possible physiological sources of 5-HT and FMRFamide are discussed. When modulators are applied to resting opener muscles, changes in membrane potential are observed. Specifically, 5-HT, MM(B), and low concentrations of MM(A) all depolarize muscle fibers. This depolarization is generally not sufficient to elicit myogenic activity in the absence of neural activity under "rest" conditions. However, if opener muscles are stretched beyond rest length, stretch- and modulator-induced depolarizations can summate and elicit contractions. This only occurs, however, if "depolarizing" modulators are applied alone. Thus other modulators (i.e., FMRFamide and high concentrations of MM(A)) hyperpolarize opener muscle fibers and can prevent depolarizing modulators from eliciting myogenic activity. All modulators tested affected parameters of motor neuron-elicited contractions of opener muscles. MM(B) and 5-HT increased contraction size over the range of concentrations tested, whereas MM(A) potentiated contractions when it was applied at lower concentrations but decreased contraction size at higher concentrations. FMRFamide decreased contraction size at all concentrations and did not affect relaxation rate. Additionally, the MMs and 5-HT increased muscle relaxation rate, decreased contraction latency, and decreased the rate at which tension was developed during motor neuron-elicited muscle contractions. Thus these modulators dramatically affect the ability of opener muscles to follow activity in the opener motor neuron B48. The possible physiological significance of these findings is discussed.

A pair of reciprocally inhibitory histaminergic sensory neurons are activated within the same phase of ingestive motor programs in Aplysia.

Previous studies have shown that each buccal ganglion in Aplysia contains two B52 neurons, one in each hemiganglion. We now show that there are two B52 neurons in a single buccal hemiganglion and four cells in an animal. We also show that the B52 neurons are histamine-immunoreactive and use reverse phase HPLC to show that the histamine-immunoreactive substance is authentic histamine. Previous studies have shown that the B52 neurons make numerous inhibitory synaptic connections with neurons active during the radula closing/retraction phase of ingestive motor programs. A computational model of the Aplysia feeding central pattern generator has, therefore, suggested that the B52 neurons play a role in terminating closing/retraction. Consistent with this idea we show that both B52 neurons fire at the beginning of radula opening/protraction. We also show that both B52 neurons are sensory neurons. They are depolarized when a flap of connective tissue adjacent to the buccal commissural arch is stretched. During ingestive feeding this is likely to occur at the peak of closing/retraction as opening/protraction begins. In the course of this study we compare the two ipsilateral B52 neurons and show that these cells are virtually indistinguishable; e.g., they use a common neurotransmitter, make the same synaptic connections, and are both sensory as well as premotor neurons. Nevertheless we show that the B52 neurons are reciprocally inhibitory. Our results, therefore, strikingly confirm theoretical predictions made by others that neurons that inhibit each other will not necessarily participate in antagonistic phases of behavior.

Effect of a serotonergic extrinsic modulatory neuron (MCC) on radula mechanoafferent function in Aplysia.

The serotonergic metacerebral cells (MCCs) and homologous neurons in related mollusks have been extensively investigated within the context of feeding. Although previous work has indicated that the MCCs exert widespread actions, MCC modulation of sensory neurons has not been identified. We characterized interactions between the MCCs and a cell that is part of a recently described group of buccal radula mechanoafferents. The cell, B21, has a peripheral process in the tissue underlying the chitinous radula [the subradula tissue (SRT)]. Previous studies have shown that B21 can fire phasically during ingestive motor programs and provide excitatory drive to the circuitry active during radula closing/retraction. We now show that activity of B21 can be modulated by serotonin (5-HT) and the MCCs. Centrally, although a slow depolarization is typically recorded in B21 as a result of MCC stimulation, this depolarization does not cause B21 to spike. It can, however, increase B21 excitability enabling a pulse that was previously subthreshold to elicit an action potential in B21. B21 is in fact rhythmically depolarized during the radula closing/retraction phase of ingestive motor programs. Thus central effects of the MCCs on radula mechanoafferent activity are only likely to be apparent while B21 is receiving input from the feeding central pattern generator. Peripherally, radula mechanoafferent neurons can be activated 1) when a mechanical stimulus is applied to the biting surface of the SRT and 2) when the SRT contracts. MCC stimulation and 5-HT modulate B21 responses to both types of stimuli. For example, MCC stimulation and low concentrations of 5-HT cause subthreshold mechanical stimuli applied to the SRT to become suprathreshold. 5-HT and MCC stimulation also enhance SRT contractility. Peripheral effects of MCC activity are also likely to be phase dependent. For example, MCC stimulation does not cause B21 to respond to peripheral stimuli with an afterdischarge. Consequently, radula mechanoafferents are likely to be activated when food is present between the radula halves during radula closing/retraction but are not likely to continue to fire as opening/protraction is initiated. In a similar vein, MCC effects on the contractility of the SRT will only be apparent when contractions are elicited by motor neuron activity. SRT motor neurons are rhythmically activated during ingestive motor programs. Thus we have shown that radula mechanoafferent activity can be modulated by the MCCs and that this modulation is likely to occur in a phase-dependent manner.

Proprioceptive input to feeding motor programs in Aplysia.

Although central pattern generators (CPGs) can produce rhythmic activity in isolation, it is now generally accepted that under physiological conditions information from the external and internal environment is incorporated into CPG-induced motor programs. Experimentally advantageous invertebrate preparations may be particularly useful for studies that seek to characterize the cellular mechanisms that make this possible. In these experiments, we study sensorimotor integration in the feeding circuitry of the mollusc Aplysia. We show that a premotor neuron with plateau properties, B51, is important for generating the radula closing/retraction phase of ingestive motor programs. When B51 is depolarized in semi-intact preparations, radula closing/retractions are enhanced. When B51 is hyperpolarized, radula closing/retractions are reduced in size. In addition to being important as a premotor interneuron, B51 is also a sensory neuron that is activated when the feeding apparatus, the radula, rotates backward. The number of centripetal spikes in B51 is increased if the resistance to backward rotation is increased. Thus, B51 is a proprioceptor that is likely to be part of a feedback loop that insures that food will be moved into the buccal cavity when difficulty is encountered. Our data suggest, therefore, that Aplysia are able to adjust feeding motor programs to accommodate the specific qualities of the food ingested because at least one of the neurons that generates the basic ingestive motor program also serves as an on-line monitor of the success of radula movements.

Synaptic mechanisms in invertebrate pattern generation.

Although individual neurons can be intrinsically oscillatory and can be network pacemakers, motor patterns are often generated in a more distributed manner. Synaptic connections with other neurons are important because they either modify the rhythm of the pacemaker cell or are essential for pattern generation in the first place. Computational studies of half-center oscillators have made much progress in describing how neurons make transitions between active and inactive phases in these simple networks. In addition to characterizing phase transitions, recent studies have described the synaptic mechanisms that are important for the initiation and maintenance of activity in half-center oscillators.

Release of peptide cotransmitters in Aplysia: regulation and functional implications.

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

Characterization of a radula opener neuromuscular system in Aplysia.

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

Multiple mechanisms for peripheral activation of the peptide-containing radula mechanoafferent neurons B21 and B22 of Aplysia.

1. Recently a cluster of sensory neurons (peptidergic radula mechanoafferents) has been identified in the buccal ganglion of Aplysia that is likely to play an important role in influencing the activity of feeding motor programs. All of the neurons of this cluster, which includes the identified cells B21 and B22, send axons via the radula nerve to a layer of tissue that lies under the chitinous radula (the subradula tissue). 2. We show that the subradula tissue has contractile properties. In the absence of the CNS, contractions of the subradula tissue are elicited if the subradula tissue is stretched. Alternatively, contractions are elicited when extracellular suction electrodes are used to stimulate buccal nerve 3 or the radula nerve. 3. Previous studies have shown that neurons of the B21/B22 cluster respond to peripherally applied mechanical stimuli. We show that these neurons are also activated when the subradula tissue contracts. Axon spikes (A spikes) can be intracellularly recorded from radula mechanoafferent neurons when contractions of the subradula tissue are elicited either by stretch or by extracellular stimulation of buccal nerve 3. 4. Mechanical stimuli that are subthreshold when applied alone elicit A spikes if they are applied while the subradula tissue is contracting. We postulate that this type of interaction may play an important role in gating sensory input to the feeding central pattern generator.

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)

A population of SCP-containing neurons in the buccal ganglion of Aplysia are radula mechanoafferents and receive excitation of central origin.

The rostral cluster of SCP-immunoreactive cells, originally identified in each buccal hemiganglion of juvenile Aplysia, was examined in mature specimens. Immunohistochemical and dye-fill experiments showed that each rostral cluster consists of approximately 40 cells. Although these neurons exhibit heterogeneity of size and shape, all cells project an axon into the radula nerve. Tracing of dye-filled cells showed that they project to the layer of tissue that lines the inner surface of the food-grasping portion of the chitinous radula. This tissue contains SCP-immunoreactive nerve fibers and varicosities in regions corresponding to the projections of dye-filled neurons. Several observations indicate that rostral cluster neurons transduce tactile stimuli applied to the radula surface: (1) each cell responds to touch of a circumscribed receptive field with a rapidly adapting burst of action potentials, (2) the evoked spikes arise abruptly from the resting potential without prepotentials, and (3) the responses persist when central and peripheral synaptic transmission is blocked in high Mg2+, low Ca2+ artificial seawater solutions. These cells, designated radula mechanoafferent (RM) neurons, do not respond to chemical stimuli including NaCl, glutamate, and seaweed extract. The highest density of receptive fields is found on the posterodorsal edges of the radula halves, areas most directly involved in grasping food. The RM neurons are electrically coupled cells, with coupling coefficients ranging from 0.006 to 0.22. They fire phasically during buccal motor programs, even in the absence of peripheral feedback from the radula or other portions of the buccal mass. In radiolabeling studies the RM cells were found to synthesize authentic SCPA and SCPB. Sensorin-A, a peptide that is localized to other Aplysia mechanoafferent neurons, was not detected immunohistochemically in these cells.

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.