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.     

Identified facilitator neurons L29 and L28 are excited by cutaneous stimuli used in dishabituation, sensitization, and classical conditioning of Aplysia

Tactile or electrical stimulation of the skin can be used to produce dishabituation, sensitization, and classical conditioning of the gill- and siphon-withdrawal reflex in Aplysia. These behavioral effects are thought to involve presynaptic facilitation at the synapses from siphon sensory neurons to gill and siphon motor neurons. Facilitation of PSPs onto the motor neurons can also be produced by intracellular stimulation of single identified neurons in the abdominal ganglion, including L29 and L28. In this paper, we further characterize L29 and L28. First, we show that they are excited by cutaneous stimuli similar to those used to produce dishabituation, sensitization, and classical conditioning and may therefore participate in mediating those behavioral effects. The results are also consistent with a possible role of L29 and L28 in higher-order features of conditioning. Second, we show that 5-HT does not mimic some of the PSPs of L29, in agreement with previous evidence that L29 is not serotonergic. Third, we present 2 types of evidence that L29 acts directly to produce facilitation of the sensory cells: (1) L29 comes into close contact with sensory cells in fluorescent double-labeling experiments, and (2) L29 produces facilitation of sensory cells in dissociated cell culture. Together with the results of the preceding paper (Mackey et al., 1989), these results indicate that facilitation of sensory cell synapses contributing to behavioral enhancement of the reflex can be produced by identified neurons that use 2 different transmitters: 5-HT (the transmitter of CB1) and the unknown transmitter of L29.     

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 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.     

Identified FMRFamide-immunoreactive neuron LPL16 in the left pleural ganglion of Aplysia produces presynaptic inhibition of siphon sensory neurons.

The gill- and siphon-withdrawal reflex of Aplysia undergoes transient inhibition following noxious stimuli such as tail shock. This behavioral inhibition appears to be due in part to transient presynaptic inhibition of the siphon sensory cells, which can be mimicked by application of the peptide FMRFamide. Although FMRFamide is widespread in the Aplysia nervous system, an FMRFamide-containing inhibitory neuron has not previously been identified. We have searched for such a neuron by combining FMRFamide immunofluorescence with fluorescent dye backfilling from the abdominal ganglion, the location of the siphon sensory cells. These methods localized a neuron in the left pleural ganglion, which we have named LPL16. LPL16 is FMRFamide immunoreactive; it is excited by tail shock; and stimulation of LPL16 produces inhibition of siphon sensory cell-to-motor cell postsynaptic potentials and narrowing of action potentials in the sensory cells in tetraethylammonium solution. These results indicate that LPL16 participates in the inhibitory effects of tail shock, and support the idea that FMRFamide plays a physiological role in the inhibition.     

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.     

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.     

Uptake of [3H]serotonin in the abdominal ganglion of Aplysia californica. Further studies on the morphological and biochemical basis of presynaptic facilitation

Sensitization of the gill-withdrawal reflex in Aplysia california is mediated, in part, by a group of identified neurons, the L29 cells, which produce presynaptic facilitation of transmitter release from siphon sensory neurons. Physiological and pharmacological studies have provided indirect evidence that the L29 cells are serotonergic. In the present study we have used the specific uptake [3H]serotonin ([3H]5-HT) and electron-microscopic autoradiography in combination with horseradish peroxidase-labeling of identified neurons to characterize the fine structure of Aplysia serotonergic terminals and to examine more directly the transmitter biochemistry of the L29 neurons. Abdominal ganglia were incubated for 2 h in 10(-6) M [3H]5-HT and thick and thin plastic sections examined with the light and electron microscope. L29 varicosities, identified by labeling with HRP, were found to accumulate [3H]5-HT. In addition, [3H]-5-HT was localized to unidentified varicosities within the neuropil as well as to vesicle-filled terminals that formed axosomatic contacts in the cortical regions of the ganglion. The processes that accumulated [3H]5-HT contained conspicuous dense core vesicles identical in morphology to those previously described for L29. Some processes were found to make contact with HRP-labeled varicosities of sensory neurons. Comparison with results obtained from ganglia exposed to [3H]5-HT in the presence of either non-radioactive 5-HT or non-radioactive dopamine indicate that the uptake process is transmitter-specific. These studies provide additional evidence that the L29 cells are serotonergic and are consistent with the notion that aminergic neurons may be preferentially involved in modulatory synaptic actions.     

Distribution of cAMP and cAMP-dependent protein kinases in Aplysia sensory neurons

Sensitization of the gill- and siphon-withdrawal reflex in Aplysia is considered a simple form of learning. Previous work has provided physiological and pharmacological evidence that cAMP-dependent protein phosphorylation within identified sensory neurons of the abdominal ganglion underlies the short-term form of this behavioral modification. Our main goal in this paper is to determine the subcellular distribution of cAMP and to measure the amounts and properties of the 2 types of subunits (regulatory and catalytic) that constitute the cAMP-dependent protein kinase. Do these biochemical parameters differ in sensory cells from those in other parts of nervous tissue? We found that the increased cAMP synthesized under conditions of sensitization is distributed in 3 compartments in the neuron: most of it is free in the cytoplasm; the remainder is bound either to cytoplasmic or to particulate proteins, which are believed to be regulatory subunits of the cAMP-dependent protein kinase. Binding of cAMP within the neurons is a measure of activation of the kinase. At rest, 17% of the binding sites in sensory cells were occupied. After brief electrical stimulation of the connective, which released endogenous transmitter, occupancy increased to 34%. This treatment increased the amount of cAMP bound to the various binding proteins differentially. The biochemical characteristics of cAMP binding were found to be the same in sensory neurons as in the rest of the nervous system but different from those in muscle. Thus, memory and learning are likely to be mediated by enzymes that are shared by other nerve cells. We found that sensory neurons have greater cAMP-dependent protein kinase activity than other neurons, however, and as a result may be more sensitive to small increases of cAMP.     

Serotonin produces long-term changes in the excitability of Aplysia sensory neurons in culture that depend on new protein synthesis

When isolated and grown in cell culture, the sensory and motor neurons of the gill withdrawal reflex of Aplysia readily form synaptic connections. Repeated exposures to 5-HT cause facilitation of the synaptic connections between co-cultured sensory and motor neurons lasting at least 24 hr. As a first step toward understanding the locus and the mechanisms underlying this long-term synaptic facilitation, we have examined the membrane excitability of the isolated presynaptic sensory neurons grown alone in dissociated cell culture. Four repeated applications of 1 microM 5-HT caused a significant increase in the excitability of sensory neurons, lasting at least 24 hr. This resembles the short-term changes in excitability seen in response to a single application of 5-HT. Unlike the short-term effect, this long-lasting change was blocked by exposure of the cells during the 5-HT treatment to 10 microM anisomycin, an inhibitor of protein synthesis. Thus, like the synaptic facilitation, the long-term change in excitability of the isolated presynaptic neurons differs from the short-term in requiring the synthesis of new protein. This finding suggests that the sensory neuron uses gene products to modulate membrane currents in its long-term response to repeated external stimuli that are not required in its short-term response to a single stimulus.     

Distribution of serotonin-immunoreactive cell bodies and processes in the abdominal ganglion of mature Aplysia

Sensitization of the gill withdrawal reflex in Aplysia californica is an elementary form of learning, in part resulting from presynaptic facilitation of the LE mechanoreceptor neurons of the abdominal ganglion. It has previously been established that either application of serotonin or direct stimulation of a group of facilitatory neurons, the L29 cells of the abdominal ganglion, can simulate the effect of physiological stimulation in producing presynaptic facilitation. Because the evidence that serotonin serves as a facilitatory transmitter was indirect, we examined the distribution of serotonin-immunoreactive fibers and cell bodies in the abdominal ganglion in order to answer two questions: (1) do the sensory neurons receive serotonergic innervation and (2) are the L29 cells serotonergic? We observed two distinctive patterns of serotonergic innervation within the ganglion, sparse and dense. The sparse pattern is correlated with a serotonin-stimulated increase in cAMP in identified target cells, while the dense innervation is not. We found a sparse distribution of serotonin-immunoreactive fibers with varicosities close to both cell bodies and processes of identified LE sensory cells. It therefore is likely that the sensory neurons do receive serotonergic innervation. We also mapped the population of serotonergic neuronal cell bodies in the ganglion, and found five clusters of neurons. Cells in one of these clusters, the identified RB neurons, had previously been shown to synthesize serotonin from tryptophan and to contain the neurotransmitter in high concentration. Identified L29 facilitator cells marked by injection with Lucifer Yellow do not contain serotonin immunoreactivity and therefore evidently are not a source of serotonergic input onto sensory cells.     

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.     

Uptake of [H]serotonin in the abdominal ganglion ofAplysia california. Further studies on the morphological and biochemical basis of presynaptic facilitation

Sensitization of the gill-withdrawal reflex inAplysia california is mediated, in part, by a group of identified neurons, the L29 cells, which produce presynaptic facilitation of transmitter release from siphon sensory neurons. Physiological and pharmacological studies have provided indirect evidence that the L29 cells are serotonergic. In the present study we have used the specific uptake [³H]serotonin ([³H]5-HT) and electron-microscopic autoradiography in combination with horseradish peroxidase-labeling of identified neurons to characterize the fine structure ofAplysia serotonergic terminals and to examine more directly the transmitter biochemistry of the L29 neurons. Abdominal ganglia were incubated for 2 h in 10⁻⁶ M [³H]5-HT and thick and thin plastic sections examined with the light and electron microscope. L29 varicosities, identified by labeling with HRP, were found to accumulate [³H]5-HT. In addition, [³H]5-HT was localized to unidentified varicosities within the neuropil as well as to vesicle-filled terminals that formed axosomatic contacts in the cortical regions of the ganglion. The processes that accumulated [³H]5-HT contained conspicuous dense core vesicles identical in morphology to those previously described for L29. Some processes were found to make contact with HRP-labeled varicosities of sensory neurons. Comparison with results obtained from ganglia exposed to [³H]5-HT in the presence of either non-radioactive 5-HT or non-radioactive dopamine indicate that the iptake process is transmitter-specific. These studies provide additional evidence that the L29 cells are serotonergic and are consistent with the notion that aminergic neurons may be preferentially involved in modulatory synaptic actions.     

Hundreds of neurons in the Aplysia abdominal ganglion are active during the gill-withdrawal reflex

A combination of optical and electrode recording methods was used to obtain an overview of the neuron activity in the Aplysia abdominal ganglion in response to a light touch to the siphon skin. Spike activity was detected in up to 150 different neurons. Habituation and sensitization of the gill-withdrawal reflex was accompanied by large changes in the number of activated neurons. It is likely that these recordings are incomplete; the actual number of activated neurons is estimated to be about 300 in the acutely sensitized preparation. While we presume that not all 300 of these neurons are involved in the gill-withdrawal reflex, the number of neurons is so large that it may be difficult to determine the role of each activated neuron with presently available experimental tools.     

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)     

Contribution of polysynaptic pathways in the mediation and plasticity of Aplysia gill and siphon withdrawal reflex: evidence for differential modulation.

The gill and siphon withdrawal (GSW) reflex of Aplysia is centrally mediated by a monosynaptic and a polysynaptic pathway between sensory and motor neurons. The first objective of this article was to evaluate quantitatively the relative importance of these two components in the mediation of the GSW reflex. We have used an artificial sea water (ASW) solution containing a high concentration of divalent cations to raise the action potential threshold of the interneurons without affecting the monosynaptic component of the reflex (2:1 ASW). Compound EPSPs induced in gill or siphon motor neurons by direct stimulation of the siphon nerve or by tactile stimulation of the siphon skin were reduced by more than 75% in 2:1 ASW. These results indicate that interneurons intercalated between sensory and motor neurons are responsible for a considerable proportion of the afferent input to the motor neurons of the reflex. The second objective of this article was to compare the modulation of the monosynaptic and polysynaptic pathways. We have evaluated their respective contribution in sensitization of the GSW reflex by testing the effects of two neuromodulators of the reflex, 5-HT and small cardioactive peptide B (SCPB). We found that these two neuromodulators have a differential action on the two components of the GSW neuronal network. The polysynaptic pathway was more facilitated than the monosynaptic pathway by the neuropeptide SCPB. By contrast, 5-HT displayed an opposite selectivity. These results suggest that the polysynaptic component of the neuronal network underlying the GSW reflex is very important for its mediation. The data also indicate that the monosynaptic and polysynaptic components of the reflex can be differentially modulated. The diversity of modulatory actions at various sites of the GSW network should be relevant for learning-associated modifications in the intact animal.     

Facilitatory transmitter causes a selective and prolonged increase in adenosine 3':5'-monophosphate in sensory neurons mediating the gill and siphon withdrawal reflex in Aplysia

Sensitization of the gill and siphon withdrawal reflex in the marine mollusc, Aplysia california, is a simple form of learning Underlying this behavioral changes is a cascade of biochemical events. The first step in this cascade is postulated to be an increase in cAMP within the sensory neurons of the abdominal ganglion. We have developed a labeling protocol with 32Pi which permits us to measure the synthesis of cAMP within a single sensory neurons. Application of serotonin for 5 min was found to triple the content of [32P]cAMP in sensory neurons. The response is specific to serotonin: dopamine, a transmitter that does not produce sensitization, did not increase cAMP. Physiological stimulation of facilitator neurons also resulted in a 3.5-fold increase of cAMP in sensory neurons but not in other cells of the ganglion. We studied the time course of the increase of cAMP in sensory cells stimulated with serotonin and found that it parallels closely the time course of the short term form of presynaptic facilitation. We also have determined the effects of transmitters on the synthesis of cAMP in other identified neurons of the ganglion. The bag cells responded specifically to serotonin. R15, which has been shown to be hyperpolarized both the serotonin and by dopamine, responded to both transmitters by increased synthesis synthesis of cAMP. Thus, the dopamine- and serotonin-sensitive cyclase can be localized to both the same and different cells. Other cells did not respond to serotonin or to dopamine, indicating that a transmitter-sensitive adenylate cyclase is a specific property and is not present in all neurons.     

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.     

Time course of structural changes at identified sensory neuron synapses during long-term sensitization in Aplysia

We have used the gill- and siphon-withdrawal reflex of Aplysia californica to explore the morphological basis of the synaptic plasticity that underlies long-term sensitization. In earlier studies (Bailey and Chen, 1983, 1988a), we described 2 classes of structural changes at identified sensory neuron synapses that occur following long-term sensitization: (1) increases in the number, size, and vesicle complement of active zones and (2) an overall increase in the total number of synaptic varicosities per sensory neuron. In the present study, we have begun to examine which of these anatomical changes might be necessary for the maintenance of long-term sensitization by exploring the time course over which they occur and, in particular, their duration relative to the persistence of the memory assessed behaviorally. Toward this end we have quantitated changes in both the total number of varicosities and their active zone morphology in single HRP-labeled sensory neurons taken from long-term sensitized and control animals at different intervals (1-2 d, 1 week, and 3 weeks) following training. We have found that long-term sensitized animals examined within 48 hr after the completion of training demonstrate an increase in the total number of varicosities per sensory neuron as well as an increase in the incidence, size, and vesicle complement of their synaptic active zones compared with control animals. The increase in the number of varicosities and active zones persists unchanged for at least 1 week, and the increase in active zone number is only partially reversed at the end of 3 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Peptidergic and serotonergic facilitation of a neuromuscular synapse in Aplysia

(1) A buccal muscle motor neuron which synthesizes the neuromodulatory small cardioactive peptides (SCPs) was identified in the buccal ganglion of Aplysia by using a combination of electrophysiological and single cell biochemical experiments. This neuron was designated B38. (2) Exogenous SCPb enhanced B38-induced contractions when perfused over the target muscle, the rostral portion of the buccal I3 muscle. SCPB potentiation of muscle contraction was associated with an increase in the excitatory junction potential (EJP) amplitude recorded from the muscle fibers, increased muscle cyclic AMP (cAMP) content, hyperpolarization of the muscle fibers, and an increase in the muscle fiber membrane conductance. Exogenous SCPB also depolarized the cell body of B38 and increased electrical coupling between the symmetrically paired B38 neurons. (3) These results suggest that the SCPs may be co-released from B38 along with an unidentified conventional neurotransmitter to homosynaptically facilitate B38 synaptic transmission by modulating presynaptic and postsynaptic components. (4) Stimulation of the identified serotonergic metacerebral neuron or perfusion of exogenous serotonin (5-HT) over the 13 muscle also potentiated B38-induced muscle contractions and EJP amplitude. Thus the B38 neuromuscular synapse represents a peripheral site of serotonergic heterosynaptic facilitation in Aplysia. (5) Presynaptic and postsynaptic serotonergic effects were qualitatively similar to those of SCPB. Serotonergic effects on muscle fiber hyperpolarization and increase in muscle fiber membrane conductance were similar in magnitude to those of SCPB but 5-HT induced a much larger increase in the EJP amplitude which was additive with that of SCPB. (6) The effect of 5-HT on the EJP amplitude was associated with inhibition of a slowly decaying component of synaptic facilitation. Concentrations of SCPB that increased the EJP were much less effective at inhibiting the slow component of facilitation. These observations indicate that 5-HT also exerted a presynaptic effect on B38 transmitter release. (7) Both 5-HT and SCPB increased muscle cAMP levels and application of forskolin mimicked many of their effects. suggesting that at least some of the postsynaptic effects were mediated by increased cAMP levels in the 13 muscle.