Development of learning and memory in Aplysia. II. Habituation and dishabituation

The defensive withdrawal reflex of the mantle organs of Aplysia californica exhibits a variety of forms of both nonassociative and associative learning, which can exist in both short- and long-term forms. In addition, the reflex can be readily elicited and quantified as soon as the effector organs (siphon and gill) emerge at their respective developmental stages. Thus, this reflex system provides a useful preparation in which to study the development of learning and memory. In the present series of experiments we investigated the development of 2 forms of nonassociative learning, habituation and dishabituation, in the siphon withdrawal component of the reflex. This reflex response could be examined throughout the juvenile life of the animal (stages 9-12) since the reflex is functionally intact as soon as the siphon emerges in stage 9 (Rankin et al., 1987). We studied the development of habituation in stages 9-12 using tactile stimuli to the siphon delivered at interstimulus intervals (ISIs) of 1, 5, 10, and 30 sec. Habituation of siphon withdrawal was evident as early as juvenile stage 9. However, it existed in an immature form: Significant habituation was produced only with a very short (1 sec) ISI. No significant habituation occurred in response to 5 or 10 sec ISIs. Approximately 4 d later, in stage 10, significant habituation occurred to both 1 and 5 sec ISIs but not to a 10 sec ISI. Finally, approximately 1-2 weeks later, in stage 11, significant habituation occurred to 1, 5, and 10 sec ISIs but not to a 30 sec ISI, whereas stage 12 juveniles and adults (stage 13) readily habituated to a 30 sec ISI. Thus, there was a systematic developmental trend in the ability of the animals to habituate: Progressively older animals were capable of habituation to stimuli presented at progressively longer intervals. The systematic development of habituation was also evident by examining the amount of habituation exhibited to comparable ISIs by animals at different developmental stages. For all 4 ISIs examined, older animals showed significantly greater habituation than younger animals. Thus, our results show that habituation is present as soon as the siphon response system emerges and that it develops progressively throughout the juvenile life of the animal. Whereas habituation was present in the earliest developmental stage we examined (stage 9), dishabituation (in response to tail shock) did not emerge until 4-7 d later, in stage 10.(ABSTRACT TRUNCATED AT 400 WORDS)     

Development of sensitization in the escape locomotion system in Aplysia

The development of several forms of nonassociative learning (habituation, dishabituation, and sensitization) has previously been examined in the gill and siphon withdrawal reflex of Aplysia. In the present study we analyzed the development of one of these forms of learning, sensitization, in a different response system in Aplysia, escape locomotion. A broad range of juvenile stages was examined: stages 10, 11, early 12, late 12, and 13 (early adult). We found that sensitization was completely absent in early developmental stages, not appearing until late stage 12. This stage of development is particularly interesting because it is at this same point that (1) sensitization first appears in the gill and siphon withdrawal reflex (Rankin and Carew, 1987), and (2) the cellular analog of sensitization first emerges in the CNS (the abdominal ganglion) of juvenile Aplysia (Nolen and Carew, 1987). The fact that sensitization emerges synchronously in the escape locomotion system and the gill withdrawal system is striking because the 2 response systems differ markedly in their intrinsic developmental timetables, response topography, and underlying neural circuitry. Thus, the emergence of sensitization in both systems at the same late stage of juvenile development suggests the possibility that a single, unified process during development may be responsible for the simultaneous expression of sensitization.     

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.     

Development of learning and memory in Aplysia. I. Functional assembly of gill and siphon withdrawal

The marine mollusc Aplysia californica provides an excellent preparation with which to examine the development of the neuronal control of behavior for 2 reasons: first, adult Aplysia exhibit a variety of behaviors that are well understood in cellular terms; and second, the development of Aplysia from embryo to adult has been studied in considerable detail. Among the best understood behaviors in Aplysia are the withdrawal responses of the mantle organs (the gill, siphon, and mantle shelf), which exhibit 2 different kinds of behaviors: "spontaneous" contractions that are part of a fixed action pattern, a respiratory pumping sequence of the mantle organs, and reflex contractions in response to tactile stimuli. We have examined the development of both of these withdrawal behaviors in juvenile stages 9-12 and found that they are functionally assembled according to different ontogenetic timetables. Spontaneous contractions. As soon as the siphon and gill emerge, in stages 9 and 10, respectively, they each show a high rate of spontaneous contraction that gradually diminishes throughout subsequent stages until it reaches the low rate typical of adults (stage 13). Since the siphon emerges first, it already exhibits a significant decline in its spontaneous activity (e.g., in stage 11) when the gill's spontaneous activity is at its highest. In addition to a developmental trend in the rate of contractions, there was also a clear developmental progression in the degree of cocontraction of the siphon and gill during spontaneous contractions. In adults, the siphon and gill show a very high degree of cocontraction during spontaneous pumping. However, in juvenile animals, there was a very low degree. Thus, it appears that the siphon and gill withdrawal components of the fixed action pattern become progressively more functionally coupled during juvenile development. Reflex contractions. As soon as the siphon and gill emerge in their respective developmental stages, they exhibit a brisk withdrawal reflex to tactile stimulation of the siphon. Moreover, at each developmental stage, reflex siphon contractions were graded as a function of stimulus intensity, as they are in the adult. Finally, throughout development tactile stimulation of the siphon invariably evoked coincident contractions of both the siphon and the gill, which is characteristic of the adult reflex. Thus, unlike the fixed action pattern that takes several weeks to mature, the defensive withdrawal reflex closely resembles the adult form as soon as the effector organs emerge during juvenile development.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

The cellular analog of sensitization in Aplysia emerges at the same time in development as behavioral sensitization

Recent studies examining the development of learning and memory in the gill and siphon withdrawal reflex of Aplysia have shown that different forms of learning emerge according to very different developmental timetables. For example, in the previous paper, Rankin and Carew (1988) showed that, whereas habituation and dishabituation emerge early in juvenile development (in stages 9 and 10, respectively), sensitization emerges at least 60 d later (in late stage 12). This developmental separation of different learning processes provides the opportunity to examine the unique contribution of specific cellular mechanisms to each form of learning. As a first step in this cellular analysis, in the present paper we have examined the development of the cellular analog of sensitization (facilitation of nondecremented EPSPs) in the identified giant neuron R2, which can serve as a monitor of the afferent input in the gill and siphon withdrawal reflex (Rayport and Camardo, 1984). We have found 2 striking parallels between the development of behavioral sensitization and the development of its cellular analog: (1) Behavioral sensitization, produced by tail shock, emerges very late in juvenile development (stage 12), and the cellular analog of sensitization (produced by activation of the tail pathway) emerges by exactly the same late juvenile stage; (2) prior to the emergence of behavioral sensitization, tail shock unexpectedly was found to produce significant reflex depression (Rankin and Carew, 1988), and prior to the emergence of the cellular analog of sensitization, activation of the tail pathway was found to produce significant depression of the synaptic input in the reflex pathway. Thus, the cellular analog of sensitization in the CNS develops and matures in close temporal register with the development of behavioral sensitization in juvenile Aplysia.     

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.     

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.     

Synaptic plasticity in vitro: cell culture of identified Aplysia neurons mediating short-term habituation and sensitization

The gill withdrawal reflex of the marine mollusk, Aplysia californica, shows habituation and sensitization, two simple forms of learning. In order to extend the cellular studies on synaptic plasticity underlying the changes in the reflex behavior, and to explore further the development of synaptic plasticity during synapse formation, we have sought to establish the neural circuit of the gill withdrawal reflex in vitro. We report here the reconstruction of the elementary gill withdrawal circuit in cell culture and find that the cells show short-term homosynaptic depression and heterosynaptic facilitation, the cellular mechanisms of habituation and sensitization, respectively.     

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.     

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.     

A behavioral analysis of habituation and sensitization of shortening in the semi-intact leech

We have previously demonstrated that the shortening reflex of the leech Hirudo medicinalis displays habituation, dishabituation, and sensitization. In this paper we demonstrate that the shortening reflex of the semi-intact animal also displays these phenomena. In the first experiment we found that the magnitude of the touch-elicited shortening reflex decreased as a result of repeated stimulations of the skin every 2 min. The second experiment examined the change in this reflex as a function of the interstimulus interval (ISI). The reflex failed to decrease when the ISI was 10 sec, but decreased significantly when the ISI was either 45 or 360 sec. Finally, in a third experiment we found that the presentation of noxious stimuli prior to habituation training prevented habituation. Thus, as we have observed previously in the intact animal, the semi-intact animal displays habituation, dishabituation, and 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.     

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.     

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)     

Effect of Feedback from Peripheral Movements on Neuron Activity in the Aplysia Abdominal Ganglion

We have made reasonably comprehensive measurements of action potential activity in the Aplysia californica abdominal ganglion to determine the amount of feedback the central nervous system (CNS) receives from a movement which it initiates. Voltage-sensitive dye measurements of action potential activity of cells in the ganglion were made during the gill-withdrawal reflex elicited by siphon stimulation. We compared recordings in two situations which differed dramatically in the amount the gill moved. In the control sea water, the gill withdrawal was normal; in low-Ca²⁺, high-Mg²⁺ sea water, the gill movement was blocked. Both the timing and the number of spikes of the individual neurons were similar in the two situations. Histograms of the summed spike activity versus time and histograms of the number of active neurons versus time in the two conditions were also similar. Finally, two numerical measures of trial-to-trial differences, a paired t -test and a measure we named fractional similarity, did not indicate larger differences between two trials in the different sea waters than two trials in the same sea water. Feedback from sensory neurons activated by the gill movement itself does not make a large contribution to the spike activity in the abdominal ganglion. Apparently the Aplysia CNS issues the command for the withdrawal and does not make adjustments for the magnitude of the actual withdrawal. It may not even receive the information necessary for such adjustments to be made. A second motivation for these experiments was to test whether removing the feedback might simplify the neuronal activity that occurs during the gill-withdrawal reflex. This did not occur.     

Habituation and dishabituation of a cleaning reflex in normal and mutant Drosophila

Upon tactile stimulation of its thoracic bristle(s), Drosophila cleans with a patterned set of leg movements the field covered by the stimulated bristles. We demonstrate that this cleaning reflex undergoes habituation and dishabituation. Repeated monotonous stimulation of the bristles by controlled air puffs leads to decrement, and finally to disappearance, of leg response. Spontaneous recovery of the response takes place in a time-dependent manner. Restoration of response can also be obtained by application of a high-frequency stimulus directed to other bristles. A mutant, rut, which is defective in learning and in adenylate cyclase activity, can habituate and dishabituate, but habituation is abnormally short-lived. As opposed to both nonassociative and associative learning paradigms used in Drosophila to date, the cleaning reflex lends itself to some aspects of cellular analysis, since single sensory neurons that mediate the input and motor neurons that mediate the behavioral output are identifiable. The modified reflex should therefore be useful in establishing the effects of single gene mutations that affect behavioral plasticity on the development and properties of identified neurons that contribute to discrete modifiable behaviors.     

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.     

An arginine vasotocin-like neuropeptide is present in the nervous system of the marine mollusc Aplysia californica

Radioimmunoassays and high pressure liquid chromotography have been used to demonstrate the presence of an arginine-vasotocin-like peptide (AVT) in the anterior ganglia of Aplysia. Previously, AVT, using similar methods, was found to be present only in vertebrates. AVT when perfused over the abdominal ganglion (10⁻⁶–10⁻¹²M) was found to increase the bursting activity of R₁₅, to decrease the bursting activity of L₃–L₆ and to increase the CNS's suppressive influence over the gill withdrawal reflex evoked by siphon stimulation. The AVT present in the nervous system of Aplysia may mediate long-term suppression of gill reflex behaviors induced by factors such as satiation and, as well, regulate the activity of certain neurosecretory neurons.