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.     

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.     

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)     

Complexities of a simple system: new lessons, old challenges and peripheral questions for the gill withdrawal reflex of Aplysia

The gill withdrawal reflex of Aplysia is generally depicted as a simple behaviour mediated by a simple neural circuit in a simple organism. Such a view has permitted a clear focus upon synapses between relatively small numbers of identified neurones, which are known to participate in the reflex and its plasticity. Ensuing research has provided some of the first and still among the most powerful explanations of the cellular underpinnings of learning and memory. In reality, however, the reflexive withdrawal of the gill and other mantle organs is anything but simple. First, the behaviour itself is complex and varies depending upon the strength of the tactile stimulus and where it is applied. In addition, over 100 central neurones are activated by stimuli, which elicit the withdrawal reflex and likely change their activities during learning (although not all of these cells necessarily contribute to the actual withdrawal response). Moreover, multiple mechanisms are activated at both presynaptic and postsynaptic sites to orchestrate the numerous modifications that underlie observed changes in synaptic efficacy. The picture becomes even more complicated when hundreds of additional peripheral neurones, which are known to participate in various aspects of the response, are also considered. Recent work has shifted attention back to these peripheral cells by suggesting that they might be the previously unidentified light touch receptors that mediate both central and peripheral components of the reflex. While daunting, the complexity of the total circuitry mediating the gill withdrawal reflex may provide yet another important lesson: even in simple systems, memory may not be localized to specific loci, but rather may be an emergent property of physiological mechanisms distributed throughout the entire circuitry.     

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.     

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)     

Habituation of the startle reflex in posttraumatic stress disorder

The authors investigated habituation of the eye-blink component of the startle reflex to repeated affectively neutral tactile and auditory stimuli in nine subjects with posttraumatic stress disorder and nine controls. Each group showed rapid habituation in both tactile and auditory modalities.     

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.     

Blink reflex in hemiplegia.

An electrophysiological study of the blink reflex was undertaken in 20 normal subjects and in 28 patients complaining of central facial palsy caused by unilateral hemispheral damage. In normal subjects, the latency, amplitude and organization of R1 and R2 responses are well known. Habituation of R2 responses occurred between 1 and 2 c/sec stimulation rate. R1 responses habituated at a higher stimulation rate (5 c/sec). In patients with unilateral hemispheral lesion, our results showed that changes in the blink reflex responses were bilateral. On the hemiplegic side the responses showed a decreased amplitude, while they were facilitated on the "normal" side. However, there was no change in latency of the two components of the reflex, on both sides. On the other hand, habituation of the late component occurred on the hemiplegic side for low stimulation rates: (0.5--1 c/sec), while on the "normal" side there was less habituation (3--4 c/sec), as compared with normal subjects. These results agree with those of experimental studies on cortical modulatory influences on brain-stem nuclei. They suggest a tactile origin of the two components of the blink reflex.     

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.     

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.     

Effects of occlusion of the thoracic aorta on habituation of the flexor withdrawal reflex in the rat

Experiments were carried out to investigate the extent to which habituation of the flexor reflex depended on mechanisms operating at spinal interneurons. An attempt was made to cause selective degeneration of interneurons in the spinal cord of the rat by subjecting the cord to a period of ischemia. Ischemia was produced by temporary occlusion of the thoracic aorta. The flexor withdrawal reflex was tested 3, 7 or 14 days after occlusion. When compared with data from control animals it was found that ischemia had resulted in both a qualitative change and a quantitative diminution in the amount of habituation which occurred during the presentation of 400 uniform stimuli. It was concluded that this impairment of the habituation process was a consequence of degeneration of interneurons which normally cause progressive inhibition of the excitatory flexor reflex pathway.     

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.     

Neurochemical modulation of sensory-motor reactivity: acoustic and tactile startle reflexes

The present review argues that the startle reflex is particularly well suited as a model system to analyze how drugs alter stimulus reactivity and reflex excitability. It then reviews all the literature to date on how drugs or lesions that are thought to alter neurochemical transmitter systems affect acoustic and/or tactile startle. Hypotheses are presented to account for how serotonin, dopamine, norepinephrine, acetylcholine, and opiates modulate startle. Effects on startle plasticity such as habituation, sensitization, and potentiation resulting from prior associative learning are also included.     

Co-localization of SCPB-like and FMRFamide-like immunoreactivities in crustacean nervous systems

A monoclonal antibody to the molluscan small cardioactive peptide SCPB and a polyclonal antibody to FMRFamide were used to localize antigens in the stomatogastric nervous system and brain of two species of Cancer. Both antibodies labeled cell bodies, axons, and neuropilar processes in the brain and in the stomatogastric nervous system. All of the SCPB immunoreactive neurons were co-labeled with antibody to FMRFamide. However, antibody to FMRFamide labeled additional neurons of the commissural ganglion and the brain that were not immunoreactive to the monoclonal SCPB antibody.     

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.     

Differential Effects of a Distant Noxious Stimulus on Hindlimb Nociceptive Withdrawal Reflexes in the Rat

Recent studies indicate that the nociceptive withdrawal reflexes to individual muscles are evoked by separate reflex pathways. The present study examines whether nociceptive withdrawal reflexes to different muscles are subject to differential supraspinal control in rats. A distant noxious stimulus was used to activate a bulbospinal system which selectively inhibits 'multireceptive' neurons (i.e. neurons receiving excitatory tactile and nociceptive inputs) in the dorsal horn of the spinal cord. Withdrawal reflexes, recorded with electromyographic techniques in single hindlimb muscles, were evoked by standardized noxious pinch. Thirty-seven rats, anaesthetized with halothane and nitrous oxide, were used. Whereas withdrawal reflexes to the extensor digitorum longus and brevis, tibialis anterior and biceps posterior muscles were strongly inhibited, reflexes to interossei muscles were potentiated during noxious pinch of the nose. Reflexes to peronei muscles were not significantly changed. The effects on the reflexes usually had an onset latency of <0.5 s and outlasted the conditioning stimulation by up to 2 s. The monosynaptic la reflex to the deep peroneal nerve, innervating dorsiflexors of the digits and ankle, was not significantly changed during noxious pinch of the nose. Hence, the inhibitory effects on the hindlimb withdrawal reflexes induced by the conditioning stimulation were presumably exerted on reflex interneurons. It is concluded that nociceptive withdrawal reflexes to different hindlimb muscles are differentially controlled by descending pathways activated by a distant noxious stimulus. The results support our previous conclusion that there are separate nociceptive withdrawal reflex pathways to different hindlimb muscles.     

Stimulation of dental pulp with simultaneous recording of the jaw opening reflex in the rat.

The processing of nociceptive information in the central nervous system has been analysed in most studies by activation of peripheral nerves. However, the limitation of this method is the simultaneous activation of noxious and inocuous fibers. Nevertheless, the stimulation of the tooth pulp is believed to activate mainly nociceptive fibers which could be the method of choice. On the other hand, the response to nociceptive activation of the dental pulp is easily quantified by the amplitude of the jaw opening reflex, a nocifensive flexion withdrawal reflex. In this protocol we describe a technique to manufacture and implant electrodes in lower incisors of the rat and a method to prepare and insert stainless steel twisted bipolar electrodes to record the electromyographic activity of both digastric muscles, in response to nociceptive dental pulp stimulation. This approach was applied in the study of the analgesic effects of the rat's striatal stimulation.     

Effects of lisuride on blink reflex habituation in Parkinson disease

The blink reflex presents a tendency to habituation (a gradual diminution of the amplitude of the response during repetitive stimulation). Electromyographic analysis of this reflex makes it possible to quantify this phenomenon. A lack of the habituation of the blink reflex is a typical feature of Parkinson disease. L-Dopa and amantadine, but not anticholinergic drugs, are able to partly reverse these abnormalities in blink reflex habituation to a normal pattern. Lisuride, a dopamine agonist with serotoninergic activity, has been recently proposed as antiparkinsonian agent. In our study we observed that lisuride has a positive effect on blink reflex habituation in Parkinson disease. A good correlation between the improvement of this electrophysiological parameter and clinical akinesia was seen. Mechanisms underlying the therapeutic effect of lisuride are complex, but this drug usually has a postsynaptic effect on D2 receptors. Our data suggest that these receptors play an important role in blink reflex habituation.     

Alterations in spontaneous miniature potential activity during habituation of a vertebrate monosynaptic pathway

Intracellular recordings of spontaneous miniature synaptic potentials were made from motoneurons of the isolated spinal cord of the frog, as a function of habituation of the monosynaptic pathway originating with lateral column fibers. The frequency and amplitude of spontaneous miniature potentials were used to assess the possibility of several proposed mechanisms underlying habituation in this system. These studies provide clear evidence in eliminating the role of transmitter depletion, incomplete vesicle filling and receptor desensitization in the habituation process occurring within this vertebrate central nervous system.