Siphon Withdrawal Reflex Of Aplysia 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.

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.

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.

Activity-Dependent Presynaptic Facilitation and Hebbian LTP Are Both Required and Interact during Classical Conditioning in Aplysia

Using a simplified preparation of the Aplysia siphon-withdrawal reflex, we previously found that associative plasticity at synapses between sensory neurons and motor neurons contributes importantly to classical conditioning of the reflex. We have now tested the roles in that plasticity of two associative cellular mechanisms: activity-dependent enhancement of presynaptic facilitation and postsynaptically induced long-term potentiation. By perturbing molecular signaling pathways in individual neurons, we have provided the most direct evidence to date that each of these mechanisms contributes to behavioral learning. In addition, our results suggest that the two mechanisms are not independent but rather interact through retrograde signaling.

Dynamic regulation of the siphon withdrawal reflex of Aplysia californica in response to changes in the ambient tactile environment.

The state of an animal's environment can be viewed as a source of information that can be used to regulate both ongoing and future behavior. The present work examined how the ambient environment can regulate the Aplysia siphon withdrawal reflex (SWR) by changing the environment between calm and turbulent. Results indicate that the SWR is dynamically regulated on the basis of variations in external conditions, so that responsiveness (measured as both reflex duration and threshold) is matched to the state of the environment. Prior exposure to a noxious stimulus (tailshock) has selective effects on this regulation, suggesting the existence of multiple regulatory mechanisms. Further, neurophysiological correlates to behavioral observations were measured in sensory and motor neurons. This will allow for a detailed cellular analysis of environmental information-processing in this system.

Modulation of cholinergic transmission in the neuronal network of the gill and siphon withdrawal reflex in Aplysia

Inhibitory interneurons are important elements of the network underlying the gill and siphon withdrawal reflex in Aplysia, and a large component of this inhibition is cholinergic. In this study, we investigated one key identified cholinergic inhibitory interneuron of the network, neuron L16, and studied some properties of its synaptic transmission and its modulation. We found that a slow inhibitory postsynaptic potential evoked in sensory neurons by L16 has two components. An earlier inhibitory postsynaptic potential component is sensitive to curare (100 μM) and has a reversal potential near the Cl − equilibrium potential (−54.5 mV). A later inhibitory postsynaptic potential component is sensitive to tetraethylammonium (0.5–1 mM); it is decreased by membrane hyperpolarization and becomes undetectable near the K + equilibrium potential (between −80 and −90 mV). Input to sensory neurons from L16 can be altered by two neuromodulators of the reflex, the small cardioactive peptide and serotonin. Small cardioactive peptide (10 μM) facilitates the connections between L16 and the sensory neurons, while serotonin (5–10 μM) inhibits them. Part of the effect of serotonin on the transmission between L16 and the sensory neurons is due to a postsynaptic mechanism, since responses to acetylcholine application in these cells are decreased by serotonin. These results indicate an additional site of synaptic plasticity in the withdrawal reflex network, the inhibitory cholinergic transmission, by two major neuromodulatory transmitters, small cardioactive peptide and serotonin.

Metaplasticity at identified inhibitory synapses in Aplysia.

Synaptic plasticity is an important feature of neural networks involved in the encoding of information. In the analysis of long-term potentiation and long-term depression, several examples have emerged in which this plasticity is itself modulated. This higher-order form of plasticity has been referred to as 'metaplasticity', a modification of synapses reflected as a change in the ability to induce or maintain plasticity. These observations raise the question of the possible advantage of regulating the intrinsic plastic properties of a synapse. The neural circuit mediating the siphon withdrawal reflex in Aplysia provides a useful network in which to examine this question directly. Inhibitory synapses in this circuit (from L30 neurons) exhibit a variety of forms of activity-dependent short-term synaptic enhancement which contribute to dynamic gain control in the siphon withdrawal reflex. Here we report that tail shock, an extrinsic modulatory input of known behavioural relevance, induces differential metaplasticity at this synapse, attenuating its ability to exhibit short-term synaptic enhancement after presynaptic activation (augmentation and post-tetanic potentiation), while leaving intact its capacity for enhancement during activation. This attenuation of inhibition at the synaptic level seems to mediate comparable attenuation of inhibitory modulation at both network and behavioural levels.

Realistic simulation of the Aplysia siphon-withdrawal reflex circuit: roles of circuit elements in producing motor output.

The circuitry underlying the Aplysia siphon-elicited siphon-withdrawal reflex has been widely used to study the cellular substrates of simple forms of learning and memory. Nonetheless, the functional roles of the different neurons and synaptic connections modified with learning have yet to be firmly established. In this study we constructed a realistic computer simulation of the best-understood component of this network to better understand how the siphon-withdrawal circuit works. We used an integrate-and-fire scheme to simulate four neuron types (LFS, L29, L30, L34) and 10 synaptic connections. Each of these circuit components was individually constructed to match the mean or typical example of its biological counterpart on the basis of group measurements of each circuit element. Once each cell and synapse was modeled, its free parameters were fixed and not subject to further manipulation. The LFS motor neurons respond to sensory input with a brief phasic burst followed by a long-lasting period of tonic firing. We found that the assembled model network responded to sensory input in a qualitatively similar fashion, suggesting that many of the interneurons important for producing the LFS firing response have now been identified. By selectively removing different circuit elements, we determined the contribution of each of the LFS firing pattern. Our first finding was that the monosynaptic sensory neuron to motor neuron pathway contributed only to the initial brief burst of the LFS firing response, whereas the polysynaptic pathway determined the overall duration of LFS firing. By making more selective deletions, we found that the circuit elements responsible for transforming brief sensory neuron discharges into long-lasting LFS firing were the slow components of the L29-LFS fast/slow excitatory postsynaptic potentials. The inhibitory L30 neurons exerted a significant braking action on the flow of excitatory information through the circuit. Interestingly, L30 lost its ability to reduce the duration of LFS firing at high stimulus intensities. This was found to be due to the intrinsic nature of L30's current-frequency relationship. Some circuit elements, including interneuron L34, and the electrical coupling between L29 and L30 were found to have little impact when subtracted from the network. These results represent a detailed dissection of the functional roles of the different elements of the siphon-elicited siphon-withdrawal circuit in Aplysia. Because many vertebrate and invertebrate circuits perform similar tasks and contain similar information processing elements, aspects of these results may be of general significance for understanding the function of motor networks. In addition, because several sites in this network store learning-related information, these results are relevant to elucidating the functional significance of the distributed storage of learned information in Aplysia.

Enhancement of sensorimotor connections by conditioning-related stimulation in Aplysia depends upon postsynaptic Ca2+.

Classical conditioning of Aplysia's siphon-withdrawal reflex is thought to be due to a presynaptic mechanism-activity-dependent presynaptic facilitation of sensorimotor connections. Recent experiments with sensorimotor synapses in dissociated cell culture, however, provide an alternative cellular mechanism for classical conditioning-Hebbian long-term potentiation (LTP) of sensorimotor connections. Induction of Hebbian LTP of these connections is mediated by activation of N-methyl-D-aspartate-related receptors and requires the postsynaptic elevation of intracellular Ca2+. To determine whether the enhancement of sensorimotor synapses during classical conditioning in Aplysia-like LTP of sensorimotor synapses in culture-also depends upon the elevation of postsynaptic Ca2+, we carried out experiments involving the cellular analog of classical conditioning of siphon withdrawal. We examined changes in the strength of monosynaptic siphon sensorimotor connections in the abdominal ganglion of Aplysia following paired presentations of sensory neuron activation and tail nerve shock. This training regimen resulted in significant enhancement of the monosynaptic sensorimotor excitatory postsynaptic potential, as compared with the sensorimotor excitatory postsynaptic potential in preparations that received only test stimulation. Infusing the motor neuron with 1,2-bis(2-aminophenoxy)ethane-N,N-N',N'-tetraacetic acid, a specific chelator of intracellular Ca2+, prior to paired stimulation training blocked this synaptic enhancement. Our results implicate a postsynaptic, possibly Hebbian, mechanism in classical conditioning in Aplysia.

Site Specificity of Short-Term and Long-Term Habituation in the Tail-Elicited Siphon Withdrawal Reflex ofAplysia

The study of habituation in animals with relatively simple nervous systems has contributed significantly to the understanding of mechanisms underlying learning and memory. Using the tail-elicited siphon withdrawal reflex of Aplysia, which is mediated in part by bilaterally symmetrical clusters of tail sensory neurons, we found that both short-term and long-term habituation can be restricted laterally, such that habituation produced by stimulation of one side of the tail does not generalize to the other side. Further experiments in this preparation revealed that long-term, laterally restricted habituation is sensitive to the temporal pattern with which stimuli are presented. We also determined that both short-term and long-term habituation can take place in a reduced behavioral preparation, and that short-term habituation can be restricted within relatively small stimulation sites located on the same side of the tail. These results provide insights into the cellular organization of habituation, and they provide a useful preparation for a cellular analysis of this basic form of learning.

A single identified interneuron gates tail-shock induced inhibition in the siphon withdrawal reflex of Aplysia

The marine mollusc Aplysia has proven very useful for a mechanistic analysis of behavioral modification. Among the stimuli used to modify the behavior of Aplysia, a noxious stimulus, tail shock, is one of the most effective. In addition to the extensively analyzed facilitatory effects of tail shock, recent work has demonstrated that it also produces marked transient inhibition in reflex responses. Here we report that functional removal (by hyperpolarization or voltage clamp) of a single inhibitory interneuron, L16, can eliminate most, if not all, of the inhibition in the siphon withdrawal reflex circuit produced by tail shock. In addition, this interneuron is strongly activated by tail shock. Finally, direct intracellular activation of L16 does not, in itself, reliably produce inhibition, suggesting that L16 plays a gating role which is necessary for the expression of inhibition in the siphon withdrawal circuit. These results support the idea that behaviorally relevant neural modulation can be gated by a small number of neurons, in this case, by a single identified cell. Moreover, they indicate that in Aplysia, as in many other systems, the modulatory effects of a noxious stimulus are often funneled through a restricted neural locus before being distributed to the circuits actually responsible for generating the behavioral output.

Cutaneous activation of the inhibitory L30 interneurons provides a mechanism for regulating adaptive gain control in the siphon withdrawal reflex of Aplysia

The functional role of inhibition in the neural network underlying the siphon withdrawal response (SWR) of Aplysia was assessed by examining a recurrent circuit comprised of identified inhibitory interneurons (L30s), and excitatory interneurons (L29s). We previously showed that activity-dependent potentiation of the L30 inhibitory synapse onto L29 can regulate the net excitatory input elicited by tactile siphon stimulation onto siphon motor neurons (LFS cells) (Fischer and Carew, 1993a). To explore the functional significance of L30 potentiated inhibition, we have examined how a behaviorally relevant stimulus that activates the L30 interneurons modulates the SWR circuit. Utilizing a reduced preparation, we show that weak tactile stimulation of the tail strongly activates the L30s, and leads to significant potentiation of the L30 synapse. Next, we demonstrate that similar weak tail stimulation produces significant inhibition of siphon tap-evoked responses in both L29 interneurons and LFS motor neurons. We further show that this form of inhibition is transient, having a time course of approximately 60 sec. Finally, we directly tested the role of the L30s in mediating this form of inhibition by hyperpolarizing two (of three) L30 interneurons during tail stimulation. L30 inactivation significantly attenuated tail stimulation-induced inhibition of siphon-evoked input to both L29 interneurons and LFS motor neurons. Based on these results, we suggest that L30-potentiated inhibition may have an important adaptive role in optimizing the signal-to-noise ratio for activation of the SWR circuit by providing stabilization of SWR responsiveness under a wide range of environmental conditions.

FMRFamide produces biphasic modulation of the LFS motor neurons in the neural circuit of the siphon withdrawal reflex of Aplysia by activating Na+ and K+ currents

The molluscan neuropeptide FMRFamide has an inhibitory effect on transmitter release from the presynaptic sensory neurons in the neural circuit for the siphon withdrawal reflex. We have explored whether FMRFamide also acts postsynaptically in motor neurons in this circuit, focusing on the LFS motor neurons. FMRFamide typically produces a biphasic response in LFS neurons: a fast excitatory response followed by a prolonged inhibitory response. We have analyzed these postsynaptic actions and compared them with the mechanism of FMRFamide's inhibition of the presynaptic sensory neurons. The transient excitatory effect of FMRFamide, which desensitizes rapidly, is due to activation of a TTX-insensitive, Na(+)-dependent inward current. The late hyperpolarizing phase of the FMRFamide response results from activation of at least two K+ currents. One component of the hyperpolarizing response is active at rest and at more hyperpolarized membrane potentials, and is blocked by 5 mM 4-aminopyridine, suggesting that it differs from the previously described FMRFamide-modulated K+ currents in the presynaptic sensory neurons. In addition, FMRFamide increases a 4-aminopyridine-insensitive K+ current. Presynaptically, FMRFamide increases K+ conductance, acting via release of arachidonic acid. In the LFS motor neurons, application of arachidonic acid mimicked the prolonged, hyperpolarizing phase of the FMRFamide response; 4-bromophenacyl bromide, an inhibitor of phospholipase A2, selectively blocked this component of the FMRFamide response. Thus, FMRFamide may act in parallel pre- and post-synaptically to inhibit the output of the siphon withdrawal reflex circuit, producing this inhibitory effect via the same second messenger in the sensory neurons and motor neurons, though a number of the K+ currents modulated in these two types of neurons are different.

Sensitization of the gill and siphon withdrawal reflex of Aplysia: multiple sites of change in the neuronal network

1. Recent studies have emphasized the major contribution of interneuronal transmission to the mediation and learning-associated modulation of the gill and siphon withdrawal (GSW) reflex of Aplysia. We wish to provide more direct support for the hypothesis that inhibitory junctions are crucial sites of plasticity. 2. In parallel experiments we investigated modulation at five major sites of synaptic transmission in the GSW network: 1) from sensory neurons to motor neurons, 2) from sensory neurons to excitatory interneurons (INTs+) 3) from INTs+ to motor neurons (MNs), 4) from inhibitory interneurons (INTs-) to INTs+, and 5) from INTs+ to INTs-. 3. While recording simultaneously from a single sensory neuron of the LE cluster, an INT+, and a MN, we found that both LE-MN and LE-INTs+ synapses were facilitated by the activation of modulator neurons by stimulation of the left pleuroabdominal connective (185 and 93%, respectively) as well as by serotonin (5-HT) (191 and 84%). Junctions of the second type were therefore less facilitated. The difference in the magnitude of facilitation at these two sites is an indication of a branch-specific, differential efficacy in the modulation of different central synapses made by a single neuron. 4. Although INT(+)-MN junctions have the capacity to display marked posttetanic potentiation, they are not significantly potentiated after connective stimulation. Sensitization of the GSW reflex is therefore not necessarily accompanied by a modification of transmission at these synapses. 5. Inhibitory transmission to INTs+ is significantly reduced by connective stimulation (36%) and by 5-HT (71%). This supports the hypothesis that a reduction of feedback inhibition into INTs+ is a major mechanism of reflex sensitization and may account for the increased evoked firing of INTs+ that is observed after connective stimulation. 6. The excitatory input to INTs- is selectively decreased by 5-HT (50%) and by the molluscan neuropeptide small cardioactive peptide B (38%). This latter effect, which could produce disinhibition of INTs+, may explain the previous observation that this peptide is able to potentiate the evoked input to MNs of the reflex at a concentration (1 microM) that fails to modify monosynaptic sensory-motor transmission. 7. These results indicate that transmission through a small neuronal network that mediates a withdrawal reflex in Aplysia may be modulated at multiple sites and by different mechanisms. These mechanisms include: 1) branch-specific facilitation of sensory neuron outputs and 2) inhibition of INT(-)-INT+ inhibitory postsynaptic potentials by endogenous modulatory neurons and by 5-HT.(ABSTRACT TRUNCATED AT 400 WORDS)

Excitatory amino acid neurotransmission at sensory-motor and interneuronal synapses of Aplysia californica

1. Although the gill and siphon withdrawal reflex of Aplysia has been used as a model system to study learning-associated changes in synaptic transmission, the identity of the neurotransmitter released by the sensory neurons and excitatory interneurons of the network mediating this behavior is still unknown. The identification of the putative neurotransmitter of these neurons should facilitate further studies of synaptic plasticity in Aplysia. 2. We report that sensory-motor transmission within this circuit is mediated through the activation of an excitatory amino acid receptor that is blocked by the non-N-methyl-D-aspartate excitatory amino acid receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 1-(4-chlorobenzoyl)-piperazine-2,3-dicarboxylic acid (CBPD). Compound postsynaptic potentials evoked in motor neurons by electrical stimulation of the siphon nerve were blocked by 92% with CNQX (75 microM) and 89% with CBPD (75 microM). 3. Simultaneous intracellular recordings were obtained from sensory neurons, excitatory interneurons, and motor neurons. Monosynaptic excitatory postsynaptic potentials (EPSPs) evoked in motor neurons by an action potential in a sensory neuron were blocked by 86% with CNQX (75 microM) and 71% with CBPD (75 microM). The two antagonists also blocked monosynaptic interneuronal EPSPs onto motor neurons by 65% and 67%, respectively. 4. Potential agonists of the synaptic receptors were puff-applied in the intact abdominal ganglion. Homocysteic acid (HCA) was found to mimic the action of the synaptically released transmitter because it strongly excites motor neurons. This effect was blocked by CNQX. Kainate and domoic acid were also effective agonists. 5. The actions of L- and D-glutamate as well as quisqualate were found to be mainly hyperpolarizing, whereas aspartate and (+/-)-amino-3-hydroxy-5-methylisoxazole-4-propionic acid had no effect. 6. Several reasons may be proposed to explain the inability of puff-applied glutamate to excite effectively the postsynaptic neurons in the intact ganglion. It is possible nonetheless that other endogenous amino acids such as HCA act as neurotransmitters at these synapses.

Activity-dependent potentiation of recurrent inhibition: a mechanism for dynamic gain control in the siphon withdrawal reflex of Aplysia

The siphon withdrawal response (SWR) of Aplysia supports several forms of learning that are under both excitatory and inhibitory control. Here we examine the role of interneuronal processing on the regulation of siphon responses, with an emphasis on the role of inhibition. We focus on the recurrent circuit formed by the excitatory interneuron L29 and the inhibitory interneuron L30, and show that this circuit provides a mechanism for use-dependent regulation of excitatory input onto siphon motor neurons. We utilized a reduced preparation in which input to the SWR circuit was elicited by taps applied to the siphon; tap-evoked EPSPs were measured in LFS siphon motor neurons. We first show that L29 is an important source of excitatory input to LFS motor neurons: voltage-clamp inactivation of a single L29 (out of five) results in a significant reduction of tap-evoked EPSPs. Next, we demonstrate that direct intracellular activation of L29, surprisingly, produces transient inhibition of evoked input to motor neurons that lasts up to 40 sec. We then provide several lines of evidence that the mechanism of L29-induced inhibition is through the recruitment and potentiation of recurrent inhibition from L30: (1) L29 activation results in reduced tap-evoked responses of other (nonactivated) L29s; (2) direct activation of L30 mimics the inhibitory effects produced by L29 activation (LFS neurons receive no direct synaptic input from L30); and (3) the L30 IPSP is significantly potentiated as a result of its own activity, whether produced directly (by L30 activation) or indirectly (through L29 activation). This IPSP potentiation has the same time course as L29-induced inhibition of motor neuron responses. Thus activity-dependent potentiation of L30 transmission can inhibit motor neuron responses, in part through inactivation of the L29 interneuronal pool. Finally, we propose that L29-L30 interactions provide a mechanism for dynamic gain control in the SWR.

Temporal asymmetry in activation of Aplysia adenylyl cyclase by calcium and transmitter may explain temporal requirements of conditioning.

Cellular experiments have suggested that during classical conditioning of the gill and siphon withdrawal reflex of Aplysia, adenylyl cyclase may serve as a molecular site of convergence for Ca2+ and serotonin (5-hydroxytryptamine; 5-HT), the cellular representations of the conditioned and unconditioned stimuli (CS and US). We explored the possible molecular basis of the behavioral requirement that the CS and US be paired within a narrow time window and in the appropriate order. To examine the temporal interactions of brief pulses of Ca2+ and 5-HT in stimulating Aplysia neural cyclase, we used a perfused-membrane cyclase assay. When brief pulses of Ca2+ and 5-HT were paired, cyclase activation depended upon the sequence of the pulses: peak cyclase activation was greater when the Ca2+ pulse immediately preceded the 5-HT pulse. Examination of the rising phase of 5-HT stimulation suggested that a Ca2+ prepulse might accelerate the onset of activation by 5-HT, without affecting the final level of activation achieved with prolonged 5-HT exposure. The observed interactions between Ca2+ and transmitter in activating cyclase could contribute importantly to the temporal requirements of conditioning for CS-US pairing.

Delayed-onset sensitization emerges after dishabituation in developing Aplysia

A recent study of the development of nonassociative learning in the siphon withdrawal reflex of Aplysia showed that dishabituation (facilitation of an habituated response) and sensitization (facilitation of a nonhabituated response) emerge according to different developmental timetables: dishabituation precedes sensitization by approximately 60 days (Rankin & Carew, 1988). Both forms of facilitation of the reflex were observed within 90 s of an electrical shock to the tail. However, more recent work by Marcus and colleagues (1988) in adult animals revealed that sensitization can have a delayed onset of 20-30 min after a strong tail shock. Since the developmental study of Rankin and Carew (1988) only tested the reflex for 10 min after tail shock, it is possible that sensitization was in fact present at earlier developmental stages, but was undetected. To examine this question, in the present study we utilized a longer (40-50 min) post-shock observation period to determine whether delayed-onset sensitization is exhibited in juvenile Aplysia, and if so, when it is expressed during development. In our first experiment, we found that Early Stage 12 juveniles (80-95 days after metamorphosis) showed significant delayed-onset sensitization 30-50 min after a strong tail shock. In a second experiment, we found that delayed-onset sensitization was absent in Stage 11 animals (20-70 days after metamorphosis). Thus delayed-onset sensitization emerges in Early Stage 12. The fact that the birthdate of delayed-onset sensitization is at least 30 days after that of dishabituation (Rankin & Carew, 1987, 1988) supports the hypothesis that these two forms of nonassociative learning may have at least partly different underlying mechanisms.

Reversal of synaptic depression by serotonin at Aplysia sensory neuron synapses involves activation of adenylyl cyclase.

Facilitation of the monosynaptic connection between siphon sensory neurons and gill and siphon motor neuron contributes to sensitization and dishabituation of the gill and siphon withdrawal reflex in Aplysia. The facilitatory transmitter serotonin (5-HT) initiates two mechanisms that act in parallel to increase transmitter release from siphon sensory neurons. 5-HT acts, at least partly through cAMP, to broaden the presynaptic action potential. 5-HT also initiates a second process that facilitates depressed sensory neuron synapses by a mechanism independent of changes in action potential duration. Recent experiments indicated that either of two protein kinases, cAMP-dependent protein kinase A and protein kinase C, are capable of effectively activating this second facilitatory mechanism, restoring synaptic transmission in depressed synapses. We have used the adenylyl cyclase inhibitor SQ 22,536 [9-(tetrahydro-2-furyl)adenine or THFA] to explore the contribution of cAMP to the reversal of synaptic depression. THFA effectively inhibited both adenylyl cyclase activity in vitro and known cyclase-mediated effects in intact sensory neurons. THFA also completely blocked facilitation of depressed synapses by 5-HT. These results suggest that adenylyl cyclase plays a critical role in the reversal of synaptic depression that contributes to dishabituation in this system.