Siphon Withdrawal Reflex Research Articles

Sleep and conditioning of the siphon withdrawal reflex in Aplysia.

Sleep is essential for memory consolidation after learning as shown in mammals and invertebrates such as bees and flies. Aplysia californica displays sleep, and sleep in this mollusk was also found to support memory for an operant conditioning task. Here, we investigated whether sleep in Aplysia is also required for memory consolidation in a simpler type of learning, i.e. the conditioning of the siphon withdrawal reflex. Two groups of animals (Wake, Sleep, each n=11) were conditioned on the siphon withdrawal reflex, with the training following a classical conditioning procedure where an electrical tail shock served as the unconditioned stimulus (US) and a tactile stimulus to the siphon as the conditioned stimulus (CS). Responses to the CS were tested before (pre-test), and 24 and 48 h after training. While Wake animals remained awake for 6 h after training, Sleep animals had undisturbed sleep. The 24 h test in both groups was combined with extinction training, i.e. the extended presentation of the CS alone over two blocks. At the 24 h test, siphon withdrawal duration in response to the CS was distinctly enhanced in both Sleep and Wake groups with no significant difference between groups, consistent with the view that consolidation of a simple conditioned reflex response does not require post-training sleep. Surprisingly, extinction training did not reverse the enhancement of responses to the CS. On the contrary, at the 48 h test, withdrawal duration in response to the CS was even further enhanced across both groups. This suggests that processes of sensitization, an even simpler non-associative type of learning, contributed to the withdrawal responses. Our study provides evidence for the hypothesis that sleep preferentially benefits consolidation of more complex learning paradigms than conditioning of simple reflexes.

Intermediate-term memory in Aplysia involves neurotrophin signaling, transcription, and DNA methylation

Long-term but not short-term memory and synaptic plasticity in many brain areas require neurotrophin signaling, transcription, and epigenetic mechanisms including DNA methylation. However, it has been difficult to relate these cellular mechanisms directly to behavior because of the immense complexity of the mammalian brain. To address that problem, we and others have examined numerically simpler systems such as the hermaphroditic marine mollusk Aplysia californica. As a further simplification, we have used a semi-intact preparation of the Aplysia siphon withdrawal reflex in which it is possible to relate cellular plasticity directly to behavioral learning. We find that inhibitors of neurotrophin signaling, transcription, and DNA methylation block sensitization and classical conditioning beginning ∼1 h after the start of training, which is in the time range of an intermediate-term stage of plasticity that combines elements of short- and long-term plasticity and may form a bridge between them. Injection of decitabine (an inhibitor of DNA methylation that may have other actions in these experiments) into an LE sensory neuron blocks the neural correlates of conditioning in the same time range. In addition, we found that both DNA and RNA methylation in the abdominal ganglion are correlated with learning in the same preparations. These results begin to suggest the functions and integration of these different molecular mechanisms during behavioral learning.

Transcriptional changes before and after forgetting of a long-term sensitization memory in Aplysia californica.

Most long-term memories are forgotten, becoming progressively less likely to be recalled. Still, some memory fragments may persist, as savings memory (easier relearning) can be detected long after recall has become impossible. What happens to a memory trace during forgetting that makes it inaccessible for recall and yet still effective to spark easier re-learning? We are addressing this question by tracking the transcriptional changes that accompany learning and then forgetting of a long-term sensitization memory in the tail-elicited siphon withdrawal reflex of Aplysia californica. First, we tracked savings memory. We found that even though recall of sensitization fades completely within 1 week of training, savings memory is still detectable at 2 weeks post training. Next, we tracked the time-course of regulation of 11 transcripts we previously identified as potentially being regulated after recall has become impossible. Remarkably, 3 transcripts still show strong regulation 2 weeks after training and an additional 4 are regulated for at least 1 week. These long-lasting changes in gene expression always begin early in the memory process, within 1 day of training. We present a synthesis of our results tracking gene expression changes accompanying sensitization and provide a testable model of how sensitization memory is forgotten.

A novel in vitro analog expressing learning-induced cellular correlates in distinct neural circuits.

When presented with noxious stimuli, Aplysia exhibits concurrent sensitization of defensive responses, such as the tail-induced siphon withdrawal reflex (TSWR) and suppression of feeding. At the cellular level, sensitization of the TSWR is accompanied by an increase in the excitability of the tail sensory neurons (TSNs) that elicit the reflex, whereas feeding suppression is accompanied by decreased excitability of B51, a decision-making neuron in the feeding neural circuit. The goal of this study was to develop an in vitro analog coexpressing the above cellular correlates. We used a reduced preparation consisting of buccal, cerebral, and pleural-pedal ganglia, which contain the neural circuits controlling feeding and the TSWR, respectively. Sensitizing stimuli were delivered in vitro by electrical stimulation of afferent nerves. When trained with sensitizing stimuli, the in vitro analog expressed concomitant increased excitability in TSNs and decreased excitability in B51, which are consistent with the occurrence of sensitization and feeding suppression induced by in vivo training. This in vitro analog expressed both short-term (15 min) and long-term (24 h) excitability changes in TSNs and B51, depending on the amount of training administered. Finally, in vitro application of serotonin increased TSN excitability without altering B51 excitability, mirroring the in vivo application of the monoamine that induces sensitization, but not feeding suppression.

Transforming growth factor β recruits persistent MAPK signaling to regulate long-term memory consolidation in Aplysia californica.

In this study, we explore the mechanistic relationship between growth factor signaling and kinase activity that supports the protein synthesis-dependent phase of long-term memory (LTM) consolidation for sensitization of Aplysia. Specifically, we examine LTM for tail shock-induced sensitization of the tail-elicited siphon withdrawal (T-SW) reflex, a form of memory that requires both (i) extracellular signal-regulated kinase (ERK1/2; MAPK) activity within identified sensory neurons (SNs) that mediate the T-SW and (ii) the activation of transforming growth factor β (TGFβ) signaling. We now report that repeated tail shocks that induce intermediate-term (ITM) and LTM for sensitization, also induce a sustained post-training phase of MAPK activity in SNs (lasting at least 1 h). We identified two mechanistically distinct phases of post-training MAPK: (i) an immediate phase that does not require ongoing protein synthesis or TGFβ signaling, and (ii) a sustained phase that requires both protein synthesis and extracellular TGFβ signaling. We find that LTM consolidation requires sustained MAPK, and is disrupted by inhibitors of protein synthesis and TGFβ signaling during the consolidation window. These results provide strong evidence that TGFβ signaling sustains MAPK activity as an essential mechanistic step for LTM consolidation.

Hyperpolarization-activated, cyclic nucleotide-gated cation channels in Aplysia: Contribution to classical conditioning

Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels are critical regulators of neuronal excitability, but less is known about their possible roles in synaptic plasticity and memory circuits. Here, we characterized the HCN gene organization, channel properties, distribution, and involvement in associative and nonassociative forms of learning in Aplysia californica. Aplysia has only one HCN gene, which codes for a channel that has many similarities to the mammalian HCN channel. The cloned acHCN gene was expressed in Xenopus oocytes, which displayed a hyperpolarization-induced inward current that was enhanced by cGMP as well as cAMP. Similarly to its homologs in other animals, acHCN is permeable to K(+) and Na(+) ions, and is selectively blocked by Cs(+) and ZD7288. We found that acHCN is predominantly expressed in inter- and motor neurons, including LFS siphon motor neurons, and therefore tested whether HCN channels are involved in simple forms of learning of the siphon-withdrawal reflex in a semiintact preparation. ZD7288 (100 μM) significantly reduced an associative form of learning (classical conditioning) but had no effect on two nonassociative forms of learning (intermediate-term sensitization and unpaired training) or baseline responses. The HCN current is enhanced by nitric oxide (NO), which may explain the postsynaptic role of NO during conditioning. HCN current in turn enhances the NMDA-like current in the motor neurons, suggesting that HCN channels contribute to conditioning through this pathway.

Associative learning in invertebrates.

This work reviews research on neural mechanisms of two types of associative learning in the marine mollusk Aplysia, classical conditioning of the gill- and siphon-withdrawal reflex and operant conditioning of feeding behavior. Basic classical conditioning is caused in part by activity-dependent facilitation at sensory neuron-motor neuron (SN-MN) synapses and involves a hybrid combination of activity-dependent presynaptic facilitation and Hebbian potentiation, which are coordinated by trans-synaptic signaling. Classical conditioning also shows several higher-order features, which might be explained by the known circuit connections in Aplysia. Operant conditioning is caused in part by a different type of mechanism, an intrinsic increase in excitability of an identified neuron in the central pattern generator (CPG) for feeding. However, for both classical and operant conditioning, adenylyl cyclase is a molecular site of convergence of the two signals that are associated. Learning in other invertebrate preparations also involves many of the same mechanisms, which may contribute to learning in vertebrates as well.

Transcriptional analysis of a whole-body form of long-term habituation in Aplysia californica.

Habituation is the simplest form of learning, but we know little about the transcriptional mechanisms that encode long-term habituation memory. A key obstacle is that habituation is relatively stimulus-specific and is thus encoded in small sets of neurons, providing poor signal/noise ratios for transcriptional analysis. To overcome this obstacle, we have developed a protocol for producing whole-body long-term habituation of the siphon-withdrawal reflex (SWR) of Aplysia californica. Specifically, we constructed a computer-controlled brushing apparatus to apply low-intensity tactile stimulation over the entire dorsal surface of Aplysia at regular intervals. We found that 3 d of training (10 rounds of stimulation/day; each round = 15 min brushing at a 10-sec ISI; 15-min rest between rounds) produces habituation with several characteristics favorable for mechanistic investigation. First, habituation is widespread, with SWR durations reduced whether the reflex is evoked by tactile stimulation to the head, tail, or the siphon. Second, long-term habituation is sensitive to the pattern of training, occurring only when brushing sessions are spaced out over 3 d rather than massed into a single session. Using a custom-designed microarray and quantitative PCR, we show that long-term habituation produces long-term up-regulation of an apparent Aplysia homolog of cornichon, a protein important for glutamate receptor trafficking. Our training paradigm provides a promising starting point for characterizing the transcriptional mechanisms of long-term habituation memory.

Reconsolidation of Long-Term Memory in Aplysia

When an animal is reminded of a prior experience and shortly afterward treated with a protein synthesis inhibitor, the consolidated memory for the experience can be disrupted; by contrast, protein synthesis inhibition without prior reminding commonly does not disrupt long-term memory [1-3]. Such results imply that the reminding triggers reconsolidation of the memory. Here, we asked whether the behavioral and synaptic changes associated with the memory for long-term sensitization (LTS) of the siphon-withdrawal reflex in the marine snail Aplysia californica [4, 5] could undergo reconsolidation. In support of this idea, we found that when sensitized animals were given abbreviated reminder sensitization training 48-96 hr after the original sensitization training, followed by treatment with the protein synthesis inhibitor anisomycin, LTS was disrupted. We also found that long-term (≥ 24 hr) facilitation (LTF) [6], which can be induced in the monosynaptic connection between Aplysia sensory and motor neurons in dissociated cell culture by multiple spaced pulses of the endogenous facilitatory transmitter serotonin (5-HT) [7, 8], could be eliminated by treating the synapses with one reminder pulse of 5-HT, followed by anisomycin, at 48 hr after the original training. Our results provide a simple model system for understanding the synaptic basis of reconsolidation.

A cellular model of memory reconsolidation involves reactivation-induced destabilization and restabilization at the sensorimotor synapse in Aplysia

The memory reconsolidation hypothesis suggests that a memory trace becomes labile after retrieval and needs to be reconsolidated before it can be stabilized. However, it is unclear from earlier studies whether the same synapses involved in encoding the memory trace are those that are destabilized and restabilized after the synaptic reactivation that accompanies memory retrieval, or whether new and different synapses are recruited. To address this issue, we studied a simple nonassociative form of memory, long-term sensitization of the gill- and siphon-withdrawal reflex in Aplysia, and its cellular analog, long-term facilitation at the sensory-to-motor neuron synapse. We found that after memory retrieval, behavioral long-term sensitization in Aplysia becomes labile via ubiquitin/proteasome-dependent protein degradation and is reconsolidated by means of de novo protein synthesis. In parallel, we found that on the cellular level, long-term facilitation at the sensory-to-motor neuron synapse that mediates long-term sensitization is also destabilized by protein degradation and is restabilized by protein synthesis after synaptic reactivation, a procedure that parallels memory retrieval or retraining evident on the behavioral level. These results provide direct evidence that the same synapses that store the long-term memory trace encoded by changes in the strength of synaptic connections critical for sensitization are disrupted and reconstructed after signal retrieval.

Protein Kinase M Maintains Long-Term Sensitization and Long-Term Facilitation inAplysia

How the brain maintains long-term memories is one of the major outstanding questions in modern neuroscience. Evidence from mammalian studies indicates that activity of a protein kinase C (PKC) isoform, protein kinase Mζ (PKMζ), plays a critical role in the maintenance of long-term memory. But the range of memories whose persistence depends on PKMζ, and the mechanisms that underlie the effect of PKMζ on long-term memory, remain obscure. Recently, a PKM isoform, known as PKM Apl III, was cloned from the nervous system of Aplysia. Here, we tested whether PKM Apl III plays a critical role in long-term memory maintenance in Aplysia. Intrahemocoel injections of the pseudosubstrate inhibitory peptide ZIP (ζ inhibitory peptide) or the PKC inhibitor chelerythrine erased the memory for long-term sensitization (LTS) of the siphon-withdrawal reflex (SWR) as late as 7 d after training. In addition, both PKM inhibitors disrupted the maintenance of long-term (≥ 24 h) facilitation (LTF) of the sensorimotor synapse, a form of synaptic plasticity previously shown to mediate LTS of the SWR. Together with previous results (Bougie et al., 2009), our results support the idea that long-term memory in Aplysia is maintained via a positive-feedback loop involving PKM Apl III-dependent protein phosphorylation. The present data extend the known role of PKM in memory maintenance to a simple and well studied type of long-term learning. Furthermore, the demonstration that PKM activity underlies the persistence of LTF of the Aplysia sensorimotor synapse, a form of synaptic plasticity amenable to rigorous cellular and molecular analyses, should facilitate efforts to understand how PKM activity maintains memory.

Behavioral adaptation of the aplysia siphon-withdrawal response is accompanied by sensory adaptation.

Behavioral adaptation is a decrease in behavioral responsiveness due to a sustained stimulus and is a key component to an organism's overall adjustment to its ambient environment. The authors examined the relationship between behavioral adaptation and sensory adaptation in the siphon-withdrawal reflex (SWR) of Aplysia californica. Sensory input to the SWR circuit was measured via en passant extracellular recordings from the siphon nerve in semi-intact preparations. The authors characterized sensory activity in response to water turbulence, an ethologically relevant stimulus that produces behavioral adaptation of the SWR. The authors found that water turbulence elicits a continuous sensory response that adapts to a low-level steady state. Consistent with behavioral measures, this sensory adaptation attenuates the evoked response to siphon taps delivered during water turbulence. Comparing trends in both behavioral and sensory adaptation revealed similar dynamics during turbulence but divergent trends during recovery. Specifically, behavioral recovery was inversely related to the duration of environmental change; recovery of sensory adaptation was not. This supports the authors' hypothesis that environment-driven behavioral adaptation reflects an interaction between sensory adaptation and additional dynamic processes.

Regulation of Behavioral and Synaptic Plasticity by Serotonin Release within Local Modulatory Fields in the CNS ofAplysia

In Aplysia, serotonergic neurons are widely activated during sensitization training, but the effects of exogenous serotonin (5-HT) on reflex circuits vary, inducing short- or long-term synaptic facilitation or synaptic inhibition, depending on the site of application. During learning, it is possible that specific spatial patterns of 5-HT release evoked by training may produce different phases of sensitization or behavioral inhibition. To test this hypothesis, we examined the modulation of the tail-induced siphon withdrawal reflex by repeated noxious stimuli applied to one of three sites: the (1) ipsilateral or (2) contralateral sides of the tail or (3) the head. Ipsilateral tail shock produced long-term sensitization, whereas contralateral tail shock induced only short-term sensitization, and head shock produced inhibition. In parallel cellular experiments, tail-nerve shock evoked large 5-HT release localized around the ipsilateral tail sensory neurons (SNs) and motor neurons (MNs) but only modest 5-HT release in the contralateral pleural-pedal ganglia and in the abdominal ganglion, in which the siphon MNs are located. Head-nerve shock, in contrast, produced only modest 5-HT release in the pleural, pedal, and abdominal ganglia. Thus, each training protocol evoked a specific pattern of 5-HT release within the CNS. In addition, we found that 5-HT released in the pleural ganglia was correlated with facilitation of SN-MN synapses; however, in the abdominal ganglion, it was associated with inhibition of the synapses between identified interneurons (L29s) and siphon MNs (LFSs). Because 5-HT differentially modulates synaptic efficacy at different synaptic sites, our data can explain how specific spatial patterns of 5-HT release in local modulatory fields can contribute to the induction of short- or long-term sensitization or to behavioral inhibition.

Latent memory for sensitization in Aplysia

In the analysis of memory it is commonly observed that, even after a memory is apparently forgotten, its latent presence can still be revealed in a subsequent learning task. Although well established on a behavioral level, the mechanisms underlying latent memory are not well understood. To begin to explore these mechanisms, we have used Aplysia, a model system that permits the simultaneous study of memory at the behavioral, cellular, and molecular levels. We first demonstrate that robust latent memory is induced by long-term sensitization training of the tail-elicited siphon withdrawal reflex. It is revealed by its ability to facilitate the subsequent induction of three mechanistically distinct temporal domains of sensitization memory: short-term, intermediate-term, and long-term memory. Under our training conditions, the latent memory persists for at least 2 d following the decay of original memory expression but appears to be gone by 4 d. Interestingly, we also find that latent memory is induced even in the absence of overt memory for the original training. These findings now permit the analysis of the cellular and molecular architecture of a common feature of learning and memory.

A Nucleolar Protein ApLLP Induces ApC/EBP Expression Required for Long-Term Synaptic Facilitation in Aplysia Neurons

In Aplysia, long-term synaptic plasticity is induced by serotonin (5-HT) or neural activity and requires gene expression. Here, we demonstrate that ApLLP, a novel nucleolus protein, is critically involved in both long-term facilitation (LTF) and behavioral sensitization. Membrane depolarization induced ApLLP expression, which activated ApC/EBP expression through a direct binding to CRE. LTF was produced by a single pulse of 5-HT 30 min after the membrane depolarization. This LTF was blocked when either ApLLP or ApC/EBP were blocked by specific antibodies. In contrast, ApLLP overexpression induced LTF in response to a single 5-HT treatment. Simultaneously, a siphon noxious stimulus (SNS) to intact Aplysia induced ApLLP and ApC/EBP expression, and single tail shock 30 min after SNS transformed short-term sensitization to long-term sensitization of siphon withdrawal reflex. These results suggest that ApLLP is an activity-dependent transcriptional activator that switches short-term facilitation to long-term facilitation.

Synaptic Facilitation and Behavioral Dishabituation in Aplysia: Dependence on Release of Ca2+ from Postsynaptic Intracellular Stores, Postsynaptic Exocytosis, and Modulation of Postsynaptic AMPA Receptor Efficacy

Sensitization and dishabituation of the defensive withdrawal reflex in Aplysia have been ascribed to presynaptic mechanisms, particularly presynaptic facilitation of transmission at sensorimotor synapses in the CNS of Aplysia. Here, we show that facilitation of sensorimotor synapses in cell culture during and after serotonin (5-HT) exposure depends on a rise in postsynaptic intracellular Ca(2+) and release of Ca(2+) from postsynaptic stores. We also provide support for the idea that postsynaptic AMPA receptor insertion mediates a component of synaptic facilitation by showing that facilitation after 5-HT offset is blocked by injecting botulinum toxin, an exocytotic inhibitor, into motor neurons before application of 5-HT. Using a reduced preparation, we extend our results to synaptic facilitation in the abdominal ganglion. We show that tail nerve shock-induced facilitation of siphon sensorimotor synapses also depends on elevated postsynaptic Ca(2+) and release of Ca(2+) from postsynaptic stores and recruits a late phase of facilitation that involves selective enhancement of the AMPA receptor-mediated synaptic response. To examine the potential role of postsynaptic exocytosis of AMPA receptors in learning in Aplysia, we test the effect of injecting botulinum toxin into siphon motor neurons on dishabituation of the siphon-withdrawal reflex. We find that postsynaptic injections of the toxin block dishabituation resulting from tail shock. Our results indicate that postsynaptic mechanisms, particularly Ca(2+)-dependent modulation of AMPA receptor trafficking, play a critical role in synaptic facilitation as well as in dishabituation and sensitization in Aplysia.

Differential role of inhibition in habituation of two independent afferent pathways to a common motor output

Many studies of the neural mechanisms of learning have focused on habituation, a simple form of learning in which a response decrements with repeated stimulation. In the siphon-elicited siphon withdrawal reflex (S-SWR) of the marine mollusk Aplysia, the prevailing view is that homosynaptic depression of primary sensory afferents underlies short-term habituation. Here we examined whether this mechanism is also utilized in habituation of the tail-elicited siphon withdrawal reflex (T-SWR), which is triggered by an independent, polysynaptic afferent pathway that converges onto the same siphon motor neurons (MNs). By using semi-intact preparations in which tail and/or siphon input to siphon MNs could be measured, we found that repeated tail stimuli administered in the presence of a reversible conduction block of the nerves downstream of the tail sensory neurons (SNs) completely abolished the induction of habituation. Subsequent retraining revealed no evidence of savings, indicating that the tail SNs and their immediate interneuronal targets are not the locus of plasticity underlying T-SWR habituation. The networks closely associated with the siphon MNs are modulated by cholinergic inhibition. We next examined the effects of network disinhibition on S-SWR and T-SWR habituation using an Ach receptor antagonist d-tubocurarine. We found that the resulting network disinhibition disrupted T-SWR, but not S-SWR, habituation. Indeed, repeated tail stimulation in the presence of d-tubocurarine resulted in an initial enhancement in responding. Lastly, we tested whether habituation of T-SWR generalized to S-SWR and found that it did not. Collectively, these data indicate that (1) unlike S-SWR, habituation of T-SWR does not involve homosynaptic depression of SNs; and (2) the sensitivity of T-SWR habituation to network disinhibition is consistent with an interneuronal plasticity mechanism that is unique to the T-SWR circuit, since it does not alter S-SWR.

Serotonergic modulation in aplysia. II. Cellular and behavioral consequences of increased serotonergic tone.

Serotonin (5-HT) plays an important role in sensitization of defensive reflexes in Aplysia and is also involved in several aspects of arousal, such as the control of locomotion and of cardiovascular tone. In the preceding paper, we showed that tail-nerve shock, a noxious stimulus that readily induces sensitization, increases the firing rate of a large number of serotonergic neurons throughout the CNS. However, the functional consequences of such an increase in serotonergic tone are still poorly understood. In this study, we examined this question by using the 5-HT precursor 5-hydroxytryptophan (5-HTP) to specifically increase 5-HT release in the CNS. Increased tonic 5-HT release after 5-HTP treatment was manifested by facilitation of sensorimotor (SN-MN) synapses, increased firing rate of serotonergic neurons in the pedal and abdominal ganglia, and enhanced 5-HT release evoked by tail-nerve shock. When 5-HTP was administered to freely moving animals, it produced a strong arousal response characterized by increased locomotion and heart rate, which was reminiscent of the defensive arousal reaction triggered by noxious stimulation such as tail-shock. In contrast, 5-HTP actually inhibited the tail-induced siphon-withdrawal reflex. It is possible that 5-HT-induced facilitation of SN-MN synapses was counteracted by inhibition of polysynaptic reflex pathways between SNs and MNs, resulting in transient behavioral inhibition of the reflex, which could favor escape locomotion and/or respiration shortly after an aversive stimulus. We conclude that a major function associated with the activation of the Aplysia serotonergic system evoked by noxious stimuli is the triggering of a defensive arousal response. It is known that tail-shock-induced serotonergic activation contributes to memory encoding at least in part by facilitating SN-MN synapses. However, this effect in isolation might not be sufficient for the behavioral expression of sensitization.

Circadian modulation of long-term sensitization in Aplysia.

As the mechanisms for learning and memory are elucidated, modulation of learning and memory becomes a central issue. We studied the modulation of learning and memory by investigating the circadian regulation of short- and long-term sensitization of the siphon withdrawal reflex in Aplysia. We found that Aplysia exhibited diurnal and circadian rhythms of long-term sensitization (LTS) with significantly greater LTS occurring when animals were trained and tested during the day relative to those trained and tested at night. In contrast to the modulation of LTS, short-term sensitization was not regulated by the circadian clock. Time of training rather than time of testing determined the circadian rhythm of LTS. Animals trained during the subjective day demonstrated LTS when tested during either the day or the night. Conversely, when animals were trained during the night, LTS was not observed when animals were tested either at night or during the day. Thus, the circadian rhythm of LTS is a rhythm in learning rather than a rhythm in recall. The threshold required to elicit siphon withdrawal and the duration of siphon withdrawal were not regulated by the circadian clock. These results indicate that the circadian oscillator exerts strong modulatory influences on one form of long-term memory in Aplysia.

Neural circuit of tail-elicited siphon withdrawal in Aplysia. II. Role of gated inhibition in differential lateralization of sensitization and dishabituation.

In the preceding report, we observed that tail-shock-induced sensitization of tail-elicited siphon withdrawal reflex (TSW) of Aplysia was expressed ipsilaterally but that dishabituation induced by an identical tail shock was expressed bilaterally. Here we examined the mechanisms of this differential lateralization. We first isolated the modulatory pathway responsible for the induction of contralateral dishabituation by making selective nerve cuts. We found that an intact pleural-abdominal connective, the descending pathway connecting the ring ganglia with the abdominal ganglion, ipsilateral to the shock was required for contralateral dishabituation. We examined whether network inhibition suppresses the contralateral effects of tail shock in nonhabituated preparations. We found that blockade of inhibitory transmission in the CNS by the nicotinic ACh inhibitor d-tubocurarine (d-TC) rendered tail shock capable of inducing bilateral sensitization. We next asked whether serotonin (5-HT), a neuromodulator released in the CNS in response to tail shock, was affected by d-TC. We found that d-TC does not alter 5-HT processes in the ring ganglia: it had no effect on the lateralized pattern of tail nerve shock-induced changes in tail sensory neuron excitability, a 5-HT-dependent process, and it did not alter tail nerve shock-evoked release of 5-HT. By contrast, d-TC enhanced 5-HT release in the abdominal ganglion. Consistent with this observation, restricting d-TC to the abdominal ganglion rendered tail nerve shock capable of producing bilateral sensitization. Together with the results of the preceding paper, our results suggest a model in which TSW sensitization and dishabituation can be dissociated both anatomically and mechanistically.