Serotonin mimics tail shock in producing transient inhibition in the siphon withdrawal reflex of Aplysia

Tail shock-induced modulation of the siphon withdrawal reflex of Aplysia has recently been shown to have a transient inhibitory component, as well as a facilitatory component. This transient behavioral inhibition is also seen in a reduced preparation in which a cellular reflection of the inhibitory process, tail shock-induced inhibition of complex EPSPs in siphon motor neurons, is observed. The biogenic amine serotonin (5-HT) is known to play a role in the facilitatory aspects of sensitization in Aplysia. The aim of this article was to examine whether 5-HT might also contribute to the inhibitory effects of tail shock in the siphon withdrawal reflex. To examine this question, we carried out two kinds of experiments. First, in the isolated abdominal ganglion, we recorded intracellularly from siphon motor neurons and examined the effects of 5-HT on (1) complex (polysynaptic) EPSPs, produced by siphon nerve stimulation, and, simultaneously, (2) monosynaptic EPSPs from siphon sensory neurons. We found that, paralleling the effects of tail shock in the reduced preparation, 5-HT produced transient inhibition of the complex EPSP; the monosynaptic EPSP was facilitated by 5-HT. Second, we examined the behavioral effects of 5-HT on siphon withdrawal in a reduced preparation. We found that 5-HT again paralleled tail shock by producing transient inhibition of the siphon withdrawal reflex. Our results suggest that, in addition to its well-established facilitatory role in reflex modulation in Aplysia, 5-HT might play an important inhibitory role, as well.     

A cellular analysis of inhibition in the siphon withdrawal reflex of Aplysia

Recent behavioral experiments examining the siphon withdrawal reflex of Aplysia have revealed inhibitory effects of strong tail shock, a stimulus commonly used as an unconditioned stimulus in studies of associative and nonassociative learning in Aplysia. We utilized a reduced preparation to perform a cellular analysis of tail shock-induced inhibition in the siphon withdrawal reflex. First, we carried out behavioral studies that showed that the reduced preparation exhibits a siphon withdrawal reflex to water jet stimuli, and that tail shock produces inhibitory behavioral effects comparable to those in the intact animal: (1) strong shock produces transient inhibition of nonhabituated responses, and (2) a habituated response is facilitated by weak shock, but not by strong shock, suggesting that increasing tail shock intensity recruits the inhibitory process that competes with facilitation of habituated reflexes. Next, we carried out cellular studies that showed that the amplitude of the complex EPSP in siphon motor neurons elicited by water jet stimuli to the siphon also exhibits the inhibitory patterns produced by tail shock: (1) the nondecremented complex EPSP (a neural correlate of a nonhabituated siphon withdrawal reflex) is significantly inhibited 90 sec after strong tail shock and recovers to preshock levels 10 min later, and (2) the decremented complex EPSP (a neural correlate of a habituated reflex) is significantly facilitated by weak shock, but is not facilitated by strong shock. In addition to the complex EPSP, we simultaneously examined the monosynaptic connection between siphon sensory neurons and siphon motor neurons. The monosynaptic EPSP does not show the pattern of inhibitory modulation by tail shock exhibited by the siphon withdrawal reflex and the complex EPSP: (1) the nondecremented monosynaptic EPSP is not inhibited 90 sec after strong shock, but tends to be above preshock levels; and (2) the decremented monosynaptic EPSP is facilitated by weak as well as strong tail shock. Our results suggest that an important component of the inhibitory process triggered by strong tail shock is mediated by neural elements presynaptic to the siphon motor neurons. Because modulation of the monosynaptic connection between identified siphon sensory and siphon motor neurons does not parallel the tail shock-induced inhibitory patterns observed in the siphon withdrawal reflex and in the complex EPSP, other synaptic connections are likely to play an important role in mediating tail shock-induced inhibition in the siphon withdrawal reflex.     

Identified serotonergic neurons LCB1 and RCB1 in the cerebral ganglia of Aplysia produce presynaptic facilitation of siphon sensory neurons

Several lines of evidence suggest that 5-HT plays a significant role in presynaptic facilitation of the siphon sensory cells contributing to dishabituation and sensitization of the gill- and siphon-withdrawal reflex in Aplysia. Most recently, Glanzman et al. (1989) found that treatment with the 5-HT neurotoxin, 5,7-DHT markedly reduced both synaptic facilitation and behavioral dishabituation. To provide more direct evidence for a role of 5-HT, we have attempted to identify individual serotonergic facilitator neurons. Hawkins (1989) used histological techniques to locate several serotonergic neurons in the ring ganglia that send axons to the abdominal ganglion and are therefore possible serotonergic facilitators. These include one neuron in the B cluster of each cerebral ganglion, which we have identified electrophysiologically and named the CB1 cells. Both glyoxylic acid histofluorescence and 5-HT immunofluorescence indicate that the CB1 neurons are serotonergic. In a semiintact preparation, the CB1 neurons respond to cutaneous stimulation which produces dishabituation and sensitization (such as tail shock) with an increase in firing, which may outlast the stimulation by 15 min. Intracellular stimulation of a CB1 neuron in a manner similar to its response to tail shock produces facilitation of the EPSPs from siphon sensory neurons to motor neurons, as well as broadening of the action potential in the sensory neurons in tetraethylammonium solution. These results strongly suggest that the identified serotonergic CB1 neurons participate in mediating presynaptic facilitation contributing to dishabituation and sensitization of the gill- and siphon-withdrawal reflex in Aplysia.     

Inhibitory neuron produces heterosynaptic inhibition of the sensory-to-motor neuron synapse in Aplysia.

We have identified an inhibitory neuron (RPL4) in the right pleural ganglion of Aplysia, which produced hyperpolarization of the sensory and motor neurons involved in the tail withdrawal reflex. Activation of RPL4 significantly reduced the amplitude of excitatory postsynaptic potentials produced in tail motor neurons by action potentials triggered in sensory neurons. This example of heterosynaptic inhibition was due, at least in part, to an increase in membrane input conductance in the motor neuron. Since the synaptic strength of the sensory-to-motor neuron connection has been associated with the strength of the tail withdrawal reflex, RPL4 may contribute to modulation of that reflex.     

Localization of potential serotonergic facilitator neurons in Aplysia by glyoxylic acid histofluorescence combined with retrograde fluorescent labeling

A variety of evidence suggests that 5-HT participates in presynaptic facilitation of the siphon sensory cells contributing to dishabituation and sensitization of the gill- and siphon-withdrawal reflex in Aplysia. Most recently, Glanzman et al. (1989) have shown that the 5-HT neurotoxin 5,7-DHT markedly reduces both the synaptic facilitation and behavioral dishabituation produced by tail shock. To provide more direct evidence for a role of 5-HT, I have used histological techniques to try to locate individual serotonergic facilitator neurons. I first used a modification of the glyoxylic acid histofluorescence technique to map serotonergic and dopaminergic neurons in the CNS of Aplysia. Intracellular fluorescent labeling combined with histofluorescence indicates that the previously identified L29 facilitator neurons are not serotonergic. Nerve transection experiments suggest that most of the perisomatic 5-HT histofluorescence in the abdominal ganglion (the location of the siphon sensory cells) comes from neurons whose cell bodies are located in the pedal or cerebral ganglia. As there are at least 500 serotonergic neurons in those ganglia, I combined retrograde fluorescent labeling with histofluorescence to identify a small subset of those neurons which send processes to the abdominal ganglion and are therefore potential serotonergic facilitators. In the following paper, Mackey et al. (1989) show that stimulation of 2 of those neurons in the cerebral ganglia (the CB1 cells) produces presynaptic facilitation of the siphon sensory cells contributing to dishabituation and sensitization of the withdrawal reflex.     

Morphology,innervation, and peripheral sensory cells of the siphon of aplysia californica

The siphon of Aplysia californica has several functions, including involvement in respiration, excretion, and defensive inking. It also provides sensory input for defensive withdrawals that have been studied extensively to examine mechanisms that underlie learning. To better understand the neuronal bases of these functions, we used immunohistochemistry to catalogue peripheral cell types and innervation of the siphon in stage 12 juveniles (chosen to allow observation of tissues in whole‐mounts). We found that the siphon nerve splits into three major branches, leading ultimately to a two‐part FMRFamide‐immunoreactive plexus and an apparently separate tyrosine hydroxylase–immunoreactive plexus. Putative sensory neurons included four distinct types of tubulin‐immunoreactive bipolar cells (one likely also tyrosine hydroxylase immunoreactive) that bore ciliated dendrites penetrating the epithelium. A fifth bipolar neuron type (tubulin‐ and FMRFamide‐immunoreactive) occurred deeper in the tissue, associated with part of the FMRFamide‐immunoreactive plexus. Our observations emphasize the structural complexity of the peripheral nervous system of the siphon, and the importance of direct tests of the various components to better understand the functioning of the entire organ, including its role in defensive withdrawal responses. J. Comp. Neurol. 523:2409–2425, 2015. © 2015 Wiley Periodicals, Inc.     

The contribution of facilitation of monosynaptic PSPs to dishabituation and sensitization of the Aplysia siphon withdrawal reflex.

To examine the relationship between synaptic plasticity and learning and memory as directly as possible, we have developed a new simplified preparation for studying the siphon-withdrawal reflex of Aplysia in which it is relatively easy to record synaptic connections between individual identified neurons during simple forms of learning. We estimated that monosynaptic EPSPs from LE siphon sensory neurons to LFS siphon motor neurons mediate approximately one-third of the reflex response measured in this preparation, which corresponds to siphon flaring in the intact animal. To investigate cellular mechanisms contributing to dishabituation and sensitization, we recorded evoked firing of LFS neurons, the siphon withdrawal produced by stimulation of an LFS neuron, the complex PSP in an LFS neuron, and the monosynaptic PSP from an "on-field" or "off-field" LE neuron to an LFS neuron during behavioral training. Unlike the simplified gill-withdrawal preparation (Cohen et al., 1997; Frost et al., 1997), in the siphon-withdrawal preparation we found no qualitative differences between the major cellular mechanisms contributing to dishabituation and sensitization, suggesting that dissociations that have been observed previously may be attributable to transient inhibition that does not occur for this component of the reflex. Furthermore, in the siphon-withdrawal preparation, all of the various cellular measures, including monosynaptic PSPs from either on-field or off-field LE neurons, changed approximately in parallel with changes in the behavior. These results provide the most direct evidence so far available that both dishabituation and sensitization involve multiple mechanisms, including heterosynaptic facilitation of sensory neuron-motor neuron PSPs.     

Serotonin and cyclic adenosine 3':5'-monophosphate modulate the potassium current in tail sensory neurons in the pleural ganglion of Aplysia

Tail sensory neurons in the pleural ganglion that mediate the afferent portion of the tail withdrawal reflex in Aplysia californica undergo heterosynaptic facilitation of transmitter release during sensitization. As in the siphon sensory neurons, the transmitter serotonin produces facilitation and also elicits a slow, decreased conductance excitatory postsynaptic potential (EPSP) in these neurons. Using voltage clamp and biochemical analyses, we have found that the slow EPSP in the pleural sensory neurons is due to a decrease in a potassium conductance identical to the S potassium current characterized in siphon sensory neurons. Like the S current, the current modulated by serotonin in the pleural sensory neurons is a non-inactivating potassium current, and it contributes to both the resting and action potentials. The current reverses in 120 mM external K+ at -20 mV, close to the predicted Nernst equilibrium potential. Intracellular cesium blocks the serotonin response, but the current is not blocked by equimolar substitution of barium for calcium, nor by 50 mM tetraethylammonium chloride. The effect of serotonin is cAMP dependent, since serotonin elevates cAMP and both cAMP injection and forskolin mimic the serotonin response. These results indicate that the mechanism associated with sensitization of the siphon-gill withdrawal reflex, a slow decreased potassium conductance, is also a component of the neuronal circuitry underlying modulation of another reflex, the tail withdrawal reflex. Therefore, two distinct populations of neurons subserving similar behavioral functions have related biophysical and biochemical properties.     

Depletion of serotonin in the nervous system of Aplysia reduces the behavioral enhancement of gill withdrawal as well as the heterosynaptic facilitation produced by tail shock

Noxious stimuli, such as electrical shocks to the animal's tail, enhance Aplysia's gill- and siphon-withdrawal reflex. Previous experimental work has indicated that this behavioral enhancement, known as dishabituation (if the reflex has been habituated) or sensitization (if it has not been habituated), might be mediated, at least in part, by the endogenous monoaminergic transmitter serotonin (5-HT). To assess 5-HT's role in dishabituation and sensitization of Aplysia withdrawal reflex, we treated Aplysia with the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT). We found that 5,7-DHT treatment significantly reduced the dishabituation of the withdrawal reflex produced by tail shock. Treatment with the neurotoxin also blocked the heterosynaptic facilitation of monosynaptic connections between siphon sensory neurons and their follower cells, which contributes to the behavioral enhancement. Analysis by high-performance liquid chromatography indicated that 5,7-DHT treatment significantly reduced 5-HT levels in the Aplysia CNS. Moreover, the neurotoxic effects of 5,7-DHT appeared to be relatively specific for serotonergic pathways. Thus, 5,7-DHT treatment did not disrupt the ability of nonserotonergic facilitatory interneurons, the L29 cells, to facilitate the connections of siphon sensory neurons. Also, 5,7-DHT reduced 5-HT-dependent, but not dopamine-dependent, histofluorescence in Aplysia central ganglia. Finally, 5,7-DHT does not reduce the levels of the facilitatory peptides SCPA and SCPB within the Aplysia CNS. Our results, together with those of Mackey et al. (1989), indicate that 5-HT plays a major role in mediating dishabituation and sensitization of Aplysia's withdrawal reflex.     

The Perfusion of the Endogenous Neuropeptide, Phe-Met-Arg-Phe-NH2 (FMRFamide) through the Gill of Aplysia Potentiates the Gill Withdrawal Reflex Evoked by Siphon Stimulation and Prevents its Habituation

Perfusion of Phe-Met-Arg-Phe-NH₂ (FMRFamide), an endogenous neuropeptide, through the gill of Aplysia significantly potentiates the amplitude of the gill withdrawal reflex evoked by tactile stimulation of the siphon with or without the central nervous system present. The potentiating effect of FMRFamide is reversible with washout. FMRFamide perfusion of the gill also prevented the gill withdrawal reflex from undergoing habituation when the tactile stimulus was presented repeatedly. Rather than habituate, the gill withdrawal reflex increased in amplitude. FMRFamide continued to prevent habituation in the absence of the parietal-visceral ganglion. We hypothesize that FMRFamide plays a role in the mediation of behavioural state primarily by affecting activity in the peripheral nervous system in the gill.     

Respiratory pumping: Neuronal control of a centrally commanded behavior in aplysia

Respiratory pumping in Aplysia californica is a relatively stereotyped behavioral pattern with three components: (1) withdrawal of gill, siphon and mantle shelf; (2) closing of parapodia; (3) heart inhibition accompanied by a decrease in vasomotor tone. This phasic behavior is triggered by a central burst-generating network of interneurons in the abdominal ganglion. During respiratory pumping, motor neurons innervating the several effector organs receive a burst of either excitatory or inhibitory synaptic input which has previously been attributed to an unidentified central command cell called Interneuron II. Several of these motor cells are also concomitantly release from tonic synaptic input, which is opposite in sign to that which they receive from Interneuron II. This tonic input has been attributed to an unidentified cell called Interneuron XI. In this paper we identify and describe some of the neurons which contribute to the burst generating network; specifically, we focus on the neurons that produce the synaptic action attributed to Interneurons II and XI. The synaptic actions attributed to Interneuron XI are produced by a single, spontaneously active neuron, cell L24. This cell is a multi-action interneuron: it produces inhibitory synaptic potentials in some follower motor neurons, excitatory synaptic potentials in other follower cells, and a conjoint excitatory-inhibitory synaptic action onto gill motor neuron L7. At low frequency, L24 is excitatory to L7. With high frequency firing of L24, the synaptic potential produced in L7 converts from excitatory to inhibitory. In contrast to Interneuron XI, which is a single cell, the synaptic potentials previously attributed to Interneuron II are actually produced by a cluster of at least 3 respiratory command cells which we call L25, L26 and L27. Each of these cells accounts for only a limited portion of the synaptic input that drives the motor neurons during respiratory pumping. For most motor neurons innervated by both the respiratory command cells and Interneuron XI, the two synaptic inputs are opposite in sign. Mutually inhibitory connections between Interneuron XI and some of the central respiratory command cells ensure that the synaptic potentials from these two sources are constrained to occur at different times. Thus, centrally commanded synaptic inhibition or excitation of these motor neurons is made more effective by simultaneous disexcitation or disinhibition of Interneuron XI input. In addition to their role in generating respiratory pumping, L24 and L26 also contribute to the mediation of the defensive gill and siphon withdrawal reflex.     

Cellular analog of differential classical conditioning in Aplysia: disruption by the NMDA receptor antagonist DL-2-amino-5-phosphonovalerate.

We previously showed that the associative enhancement of Aplysia siphon sensorimotor synapses in a cellular analog of classical conditioning is disrupted by infusing the Ca(2+) chelator 1, 2-bis(2-aminophenoxy)ethane-N,N-N',N'-tetraacetic acid into the postsynaptic motor neuron before training or by training in the presence of the NMDA receptor antagonist DL-2-amino-5-phosphonovalerate (APV). Our earlier experiments with APV used a nondifferential training protocol, in which different preparations were used for associative and nonassociative training. In the present experiments we extended our investigation of the role of NMDA receptor type potentiation in learning in Aplysia to differential conditioning. A cellular analog of differential conditioning was performed with a reduced preparation that consisted of the CNS plus two pedal nerves. A siphon motor neuron and two siphon sensory neurons, both of which were presynaptically connected to the motor neuron, were impaled with sharp microelectrodes. One sensorimotor synapse received paired stimulation with a conditioned stimulus (brief activation of a single sensory neuron) and an unconditioned stimulus (pedal nerve shock), whereas the other sensorimotor synapse received unpaired stimulation. Training in normal artificial seawater (ASW) resulted in significant differential enhancement of synapses that received the paired stimulation. Training in APV blocked this differential synaptic enhancement. A comparison of the present data with the data from earlier experiments that used nondifferential training is consistent with the possibility that differential training comprises competition between the presynaptic sensory neurons. Synaptic competition may contribute significantly to the associative effect of paired stimulation in the differential training paradigm.     

Heterosynaptic facilitation and behavioral sensitization are inhibited by lowering endogenous cAMP in Aplysia

An adenylate cyclase inhibitor, RMI 12330A, is able to depress cAMP synthesis stimulated by serotonin in the abdominal ganglion of Aplysia depilans and punctata. This substance reversibly blocked the heterosynaptic facilitation, induced by activation of serotonergic pathways, of the EPSP recorded from L7 motoneuron in abdominal ganglion after electrical stimulation of the siphon nerve. RMI 12330A, injected into whole unrestrained animals, inhibited the short-term dishabituation of the siphon withdrawal reflex. These findings demonstrate that the increase of endogenous cAMP in the sensory neurons mediating the gill and siphon withdrawal reflex is an essential step in the mechanism of potentiation of the transmitter output underlying heterosynaptic facilitation and short-term behavioral sensitization.     

Identification of Aplysia neurons containing immunoreactive FMRFamide

Electrophysiological and immunocytochemical techniques were used in the abdominal ganglion of Aplysia to identify neurons containing immunoreactive FMRFamide. Large numbers of neurons were immunoreactive for FMRFamide, including R2, L2, L3, L4, L5, L6, 2 cells tentatively identified as L12 and L13, and a previously unidentified cluster on the ventral surface of the right lower quadrant. There was also heavy labelling of fibers, often with beaded varicosities, throughout the neuropil, the cell layers, and the sheath overlying the ganglion. This data provides further evidence that FMRFamide is an important neurotransmitter in Aplysia. The demonstration of immunoreactive FMRFamide in the giant cholinergic neurons R2 and LP1(1) suggests that these well-studied and experimentally convenient cells use acetylcholine and an FMRFamide-like peptide as cotransmitters.     

Development of learning and memory in Aplysia. III. Central neuronal correlates

The defensive withdrawal reflex of the mantle organs of Aplysia californica has 2 major components, siphon withdrawal and gill withdrawal. In the previous paper of this series (Rankin and Carew, 1987), the development of 2 forms of nonassociative learning, habituation and dishabituation, was examined in the siphon withdrawal component of the reflex. In the present study we examined these same forms of learning in the gill withdrawal component of the reflex. The purpose of these experiments was 2-fold: to examine the development of learning in the other major component of the reflex; and to establish preparations in which it is possible to carry out a cellular analysis of the development of learning in the CNS. We first established that the gill withdrawal reflex in intact animals exhibited significant habituation in response to repeated tactile stimulation of the siphon and significant dishabituation in response to tail shock. We next determined the contribution of the CNS to the gill withdrawal reflex by surgically removing the abdominal ganglion from intact animals. Using the same stimulus intensity (4 mg) that produced habituation in the previous experiments, we found that the CNS accounted for approximately 95% of the reflex. Finally, we developed 2 preparations that allowed us to relate behavioral observations of learning directly to neural plasticity exhibited in the CNS. In a semi-intact preparation gill withdrawal was behaviorally measured as in the intact animal, but tactile stimulation of the siphon (to produce habituation) and shock to the tail (to produce dishabituation) were replaced by electrical stimulation of the siphon nerve and left connective, respectively. Stimulation parameters were matched to produce behavioral responses comparable with those in the intact animal. In an isolated CNS preparation the same nerve stimuli were used as in the semi-intact preparation, but the response measure used was the evoked neural discharge recorded in an efferent nerve innervating the gill. Both preparations exhibited response decrement and facilitation that was quantitatively as well as qualitatively similar to that observed in intact animals, indicating that 2 simple forms of learning exhibited by the gill withdrawal reflex in juvenile Aplysia can be localized to neural circuits within the abdominal ganglion.     

FMRFamide peptides in Helisoma: identification and physiological actions at a peripheral synapse

Previous reports have demonstrated powerful neuromodulatory actions of the molluscan tetrapeptide FMRFamide in both the central and peripheral nervous systems of the freshwater snail Helisoma. The present study was designed to examine both the nature of the FMRFamide-like peptides in Helisoma and to define their physiological actions at a peripheral synapse. We report that, as determined by HPLC/RIA and mass spectrometry, Helisoma contains both FMRFamide and 2 of its analogs, FLRFamide and GDPFLRFamide. Whereas whole animals contain about 100 pmol/gm of these peptides, they were enriched in the nervous system (3000 pmol/gm) and in a peripheral target organ, the salivary glands (500 pmol/gm). For histochemical and physiological studies we examined the salivary glands, which are known to be innervated by neuron 4 of the buccal ganglion. We confirmed the presence of FMRFamide-like fibers on the salivary gland by immunohistochemistry using a polyclonal antiserum. These fibers appear to be largely derived from somata located in the central ring ganglia. For physiological tests we examined the neuron 4-gland synapse, at which presynaptic action potentials normally evoke a suprathreshold EPSP in gland cells. Bath application of FMRFamide, FLRFamide, or GDPFLRFamide at micromolar concentration to a buccal ganglia/salivary gland preparation completely suppressed spontaneous rhythmic activity. The sites of action of these peptides were examined by iontophoretic application of FMRFamide to neuron 4 or the salivary gland. Application of the peptide to the soma of neuron 4 caused a hyperpolarization that suppressed spontaneously generated action potentials. When applied to the salivary gland, FMRFamide caused a hyperpolarization that reduced the EPSPs evoked by neuron 4 to below spike threshold. The latter observation implies a postsynaptic locus of action for FMRFamide, and this possibility was tested by direct depolarization of the gland with iontophoresis of ACh (the putative transmitter of neuron 4). Such depolarizations were also reduced by FMRFamide. We conclude that Helisoma contains FMRFamide and 2 of its analogs, these peptides being enriched in the nervous system and salivary glands. Furthermore, these peptides can suppress activation of the salivary glands by actions both directly on gland cells and on the effector neuron.     

Differential effects of serotonin, FMRFamide, and small cardioactive peptide on multiple, distributed processes modulating sensorimotor synaptic transmission in Aplysia.

At least two processes contribute to the modulation by 5-HT of the connections between sensory neurons and motor neurons in Aplysia. The first involves broadening of the presynaptic spike through modulation of 5-HT-sensitive K+ channels that leads to elevated levels of intracellular Ca2+ and increased release of transmitter. A second process (or set of processes) apparently accounts for the amount of facilitation not produced by presynaptic spike broadening. This spike duration-independent (SDI) process is particularly prominent in depressed synapses. We used a protocol in which spikes were prebroadened into a range of durations in which further spike broadening by itself has little or no effect on facilitation of the EPSP.5-HT produced pronounced facilitation in depressed synapses under these conditions. Another modulatory agent, small cardioactive peptide (SCPb), also broadened spikes in sensory neurons but did not produce facilitation comparable to that produced by 5-HT. These results indicate that 5-HT activates the SDI process whereas SCPb fails to do the same. A 5 min preexposure to the modulatory peptide FMRFamide inhibited 5-HT-induced activation of the SDI process, whereas a 1 min preexposure did not. Another process(es) that may modulate synaptic efficacy in sensorimotor synapses involves changes in the properties of the motor (follower) neuron, such as input resistance. FMRFamide decreased the input resistance of postsynaptic neurons. This action could contribute to the effects of FMRFamide when administered alone, but it did not appear to be responsible for the inhibitory action of FMRFamide on 5-HT-induced facilitation. Neither 5-HT nor SCPb had a clear effect on input resistance. The actions of these three agents, therefore, seem to be differentially distributed among various pre- and postsynaptic processes involved in the modulation of synaptic transmission.     

SCP-containing R20 neurons modulate respiratory pumping in Aplysia

Respiratory pumping in Aplysia consists of transient, synchronous pumping actions of the gill, siphon, mantle shelf, and parapodia. This behavior has previously been shown to be driven by a network of coupled interneurons in the abdominal ganglion, the R25 and the L25 cells. We describe here a pair of electrically coupled cells, the R20 cells, which when active can initiate respiratory pumping or increase its spontaneous rate of occurrence. This action is mediated by a slow, long-lasting excitation of the endogenous burst mechanism of the cells in the R25/L25 network. The R20 cells, which are located in the abdominal ganglion, also make slow inhibitory connections to the RB cells and to the RG cells in that ganglion, and to the gill motoneurons in the branchial ganglion. The R20 cells are immunoreactive to SCPB, a molluscan neuropeptide. Biochemical purification studies demonstrate that each of the R20 cells synthesizes not only SCPB, but also SCPA, a closely related molecule known to be encoded by the same gene as SCPB. The R20 cells also synthesize in abundance several other low-molecular-weight, methionine-containing peptides. The excitatory actions of the R20 cells on the R25/L25 network are mimicked by SCPA and SCPB. However, the inhibitory actions of the R20 cells on the RB cells, the RG cells, and on the cells of the branchial ganglion are not mimicked by the SCPs. Thus, the data support the hypothesis that the R20 cells release SCPA and SCPB and at least one other unidentified transmitter.     

Innervation of the kidney of Aplysia by L10, the LUQ cells, and an identified peripheral motoneuron

The purpose of this study was to begin to describe the neural circuit within the abdominal ganglion that modulates renal functioning in Aplysia. We found that the previously described cholinergic neuron L10 and peptidergic left upper quadrant (LUQ) neurons have important roles in the control of the kidney. Cell L10 and a subset of the LUQ cells branch extensively within the kidney and send major processes to the renal pore, a sphincter that controls the efflux of urine. The renal pore has circular (closer) and radial (opener) muscle fibers that act as antagonists. Embedded within the wall of the renal pore is a newly identified peripheral neuron, RPO, which is a renal pore opener motoneuron. L10 activity causes opening of the renal pore by directly exciting pore opener muscle, inhibiting closer muscle, and exciting RPO. When RPO is active, it generates synchronous, discrete twitches in the opener muscle fibers. The action potentials recorded in RPO exhibit pronounced broadening at physiological rates of firing. LUQ cells that project to the renal pore cause it to close, and they antagonize the opening generated by an L10 burst. The pore closing caused by the LUQ cells is mediated in part by heterosynaptic inhibition of the L10 to RPO excitatory connection. The previously described central inhibitory connections from L10 to the LUQ cells ensure that these 2 classes of antagonists fire out of phase with each other. Our data, along with those from earlier studies demonstrating that L10 plays an important role in controlling the circulatory system, suggest that L10 and the LUQ cells modulate various aspects of renal function in Aplysia, including filtration and micturition.     

Dishabituation and sensitization emerge as separate processes during development in Aplysia

Until recently, dishabituation and sensitization have commonly been considered to reflect a unitary process: Sensitization refers to a general facilitation produced by strong or noxious stimuli that enhances subsequent responding; dishabituation has been thought to represent a special instance of sensitization in which the facilitation is simply superimposed on a habituated response level. The unitary process hypothesis was based on the observation that both decremented and nondecremented responses are facilitated by a common noxious or strong stimulus. However, this observation does not rule out the possibility that dishabituation and sensitization could reflect separate processes that are activated in parallel by a strong stimulus. Recent cellular experiments by Hochner et al. (1986) suggest that this, in fact, occurs in the sensory neurons of the gill withdrawal reflex in Aplysia. A developmental analysis of learning in the marine mollusc Aplysia permits a direct behavioral test of this hypothesis. If dishabituation and sensitization reflect a unitary process then they should emerge at the same time ontogenetically. On the other hand, if they reflect different processes, then they might emerge according to different ontogenetic timetables. In the present study we examined the temporal emergence of dishabituation and sensitization in the defensive siphon withdrawal reflex in 3 stages of juvenile Aplysia: stage 11, early stage 12, and late stage 12. Animals received one of 2 kinds of training: Dishabituation training, in which the effect of strong tail shock on habituated responses were observed, and Sensitization training, in which the effect of strong tail shock on nondecremented responses was observed. We found that, while dishabituation was present in all stages examined, sensitization did not emerge until several weeks later, in late stage 12. These results were confirmed and extended in a group of animals that were tested twice: first in stage 11, when they showed no sensitization, and again 13 weeks later, in late stage 12, when they then showed significant sensitization. Our analysis of nondecremented responses prior to the emergence of sensitization also revealed an unexpected inhibitory component of tail shock that produces reflex depression. Moreover, there was a clear progression in the net effects of tail shock during development: reflex depression was produced in stages 11 and early stage 12, followed by a transition to reflex facilitation (sensitization) in late stage 12. Finally, when sensitization emerged in late stage 12, the process of dishabituation showed a significant increase compared with previous developmental stages.(ABSTRACT TRUNCATED AT 400 WORDS)