Gill-withdrawal Reflex In Aplysia Research Articles

Serotonin Induces Structural Plasticity of Both Extrinsic Modulating and Intrinsic Mediating Circuits In Vitro in Aplysia Californica

Long-term sensitization of the gill withdrawal reflex in Aplysia requires heterosynaptic, modulatory input that is mediated in part by the growth of new synaptic connections between sensory neurons and their follower cells (intrinsic mediating circuit). Whether modulatory interneurons (the extrinsic modulatory circuit) also display learning-related structural synaptic plasticity remains unknown. To test this idea, we added a bona fide serotonergic modulatory neuron, the metacerebral cell (MCC), to sensory-motor neuron co-cultures and examined the modulating presynaptic varicosities of MCCs before and after repeated pulses of serotonin (5-HT) that induced long-term facilitation (LTF). We observed robust growth of new serotonergic varicosities that were positive for serotonin and capable of synaptic recycling. Our findings demonstrate that, in addition to structural changes in the intrinsic mediating circuit, there are also significant learning-related structural changes in the extrinsic modulating circuit, and these changes might provide a cellular mechanism for savings and for spread of memory.

Synapse Formation Activates a Transcriptional Program for Persistent Enhancement in the Bi-directional Transport of Mitochondria

Mechanisms that regulate the bi-directional transport of mitochondria in neurons for maintaining functional synaptic connections are poorly understood. Here, we show that in the pre-synaptic sensory neurons of the Aplysia gill withdrawal reflex, the formation of functional synapses leads to persistent enhancement in the flux of bi-directional mitochondrial transport. In the absence of a functional synapse, activation of cAMP signaling is sufficient to enhance bi-directional transport in sensory neurons. Furthermore, persistent enhancement in transport does not depend on NMDA and AMPA receptor signaling nor signaling from the post-synaptic neuronal cell body, but it is dependent on transcription and protein synthesis in the pre-synaptic neuron. We identified ∼4,000 differentially enriched transcripts in pre-synaptic neurons, suggesting a long-term change in the transcriptional program produced by synapse formation. These results provide insights into the regulation of bi-directional mitochondrial transport for synapse maintenance.

Huntingtin is critical both pre- and postsynaptically for long-term learning-related synaptic plasticity in Aplysia.

Patients with Huntington’s disease exhibit memory and cognitive deficits many years before manifesting motor disturbances. Similarly, several studies have shown that deficits in long-term synaptic plasticity, a cellular basis of memory formation and storage, occur well before motor disturbances in the hippocampus of the transgenic mouse models of Huntington’s disease. The autosomal dominant inheritance pattern of Huntington’s disease suggests the importance of the mutant protein, huntingtin, in pathogenesis of Huntington’s disease, but wild type huntingtin also has been shown to be important for neuronal functions such as axonal transport. Yet, the role of wild type huntingtin in long-term synaptic plasticity has not been investigated in detail. We identified a huntingtin homolog in the marine snail Aplysia, and find that similar to the expression pattern in mammalian brain, huntingtin is widely expressed in neurons and glial cells. Importantly the expression of mRNAs of huntingtin is upregulated by repeated applications of serotonin, a modulatory transmitter released during learning in Aplysia. Furthermore, we find that huntingtin expression levels are critical, not only in presynaptic sensory neurons, but also in the postsynaptic motor neurons for serotonin-induced long-term facilitation at the sensory-to-motor neuron synapse of the Aplysia gill-withdrawal reflex. These results suggest a key role for huntingtin in long-term memory storage.

Neurexin-Neuroligin Transsynaptic Interaction Mediates Learning-Related Synaptic Remodeling and Long-Term Facilitation in Aplysia

Neurexin and neuroligin, which undergo heterophilic interactions with each other at the synapse, are mutated in some patients with autism spectrum disorder, a set of disorders characterized by deficits in social and emotional learning. We have explored the role of neurexin and neuroligin at sensory-to-motor neuron synapses of the gill-withdrawal reflex in Aplysia, which undergoes sensitization, a simple form of learned fear. We find that depleting neurexin in the presynaptic sensory neuron or neuroligin in the postsynaptic motor neuron abolishes both long-term facilitation and the associated presynaptic growth induced by repeated pulses of serotonin. Moreover, introduction into the motor neuron of the R451C mutation of neuroligin-3 linked to autism spectrum disorder blocks both intermediate-term and long-term facilitation. Our results suggest that activity-dependent regulation of the neurexin-neuroligin interaction may govern transsynaptic signaling required for the storage of long-term memory, including emotional memory that may be impaired in autism spectrum disorder.

Imaging and analysis of evoked excitatory-postsynaptic-calcium-transients by individual presynaptic-boutons of cultured Aplysia sensorimotor synapse

The use of the sensory–motor (SN-MN) synapse of the Aplysia gill withdrawal reflex has contributed immensely to the understanding of synaptic transmission, learning and memory acquisition processes. Whereas the majority of the studies focused on analysis of the presynaptic mechanisms, recent studies indicated that as in mammalian synapses, long term potentiation (LTP) formed by Aplysia SN-MN synapse depends on elevation of the postsynaptic free intracellular calcium concentration ([Ca2+]i). Consistently, injection of the fast calcium chelator BAPTA to the MN prevents the formation of serotonin-induced LTP. Nevertheless, currently there are no published reports that directly examine and document whether evoked synaptic transmission is associated with transient increase in the postsynaptic [Ca2+]i. In the present study we imaged, for the first time, alterations in the postsynaptic [Ca2+]i in response to presynaptic stimulation and analyzed the underlying mechanisms.Using live imaging of the postsynaptic [Ca2+]i while monitoring the EPSP, we found that evoked transmitter release generates excitatory postsynaptic calcium concentration transients (EPSCaTs) by two mechanisms: (a) activation of DNQX-sensitive postsynaptic receptors-gated calcium influx and (b) calcium influx through nitrendipine-sensitive voltage-gated calcium channels (VGCCs). Concomitant confocal imaging of presynaptic boutons and EPSCaTs revealed that approximately 86% of the presynaptic boutons are associated with functional synapses.

Fly Memory: A Mushroom Body Story in Parts

Recent studies on the compartmentalization of fly mushroom bodies show that learning and memory in Drosophila are not as simple as might be expected for an organism with such a tiny brain.

Motor Control in a Drosophila Taste Circuit

Tastes elicit innate behaviors critical for directing animals to ingest nutritious substances and reject toxic compounds, but the neural basis of these behaviors is not understood. Here, we use a neural silencing screen to identify neurons required for a simple Drosophila taste behavior and characterize a neural population that controls a specific subprogram of this behavior. By silencing and activating subsets of the defined cell population, we identify the neurons involved in the taste behavior as a pair of motor neurons located in the subesophageal ganglion (SOG). The motor neurons are activated by sugar stimulation of gustatory neurons and inhibited by bitter compounds; however, experiments utilizing split-GFP detect no direct connections between the motor neurons and primary sensory neurons, indicating that further study will be necessary to elucidate the circuitry bridging these populations. Combined, these results provide a general strategy and a valuable starting point for future taste circuit analysis.

Nuclear Translocation of CAM-Associated Protein Activates Transcription for Long-Term Facilitation in Aplysia

Repeated pulses of serotonin (5-HT) induce long-term facilitation (LTF) of the synapses between sensory and motor neurons of the gill-withdrawal reflex in Aplysia. To explore how apCAM downregulation at the plasma membrane and CREB-mediated transcription in the nucleus, both of which are required for the formation of LTF, might relate to each other, we cloned an apCAM-associated protein (CAMAP) by yeast two-hybrid screening. We found that 5-HT signaling at the synapse activates PKA which in turn phosphorylates CAMAP to induce the dissociation of CAMAP from apCAM and the subsequent translocation of CAMAP into the nucleus of sensory neurons. In the nucleus, CAMAP acts as a transcriptional coactivator for CREB1 and is essential for the activation of ApC/EBP required for the initiation of LTF. Combined, our data suggest that CAMAP is a retrograde signaling component that translocates from activated synapses to the nucleus during synapse-specific LTF.

Enhancement of Memory-Related Long-Term Facilitation by ApAF, a Novel Transcription Factor that Acts Downstream from Both CREB1 and CREB2

The memory for sensitization of the gill withdrawal reflex in Aplysia is reflected in facilitation of the monosynaptic connection between the sensory and motor neurons of the reflex. The switch from short- to long-term facilitation requires activation of CREB1, derepression of ApCREB2, and induction of ApC/EBP. In search for genes that act downstream from CREB1, we have identified a transcription activator, ApAF, which is stimulated by protein kinase A and can dimerize with both ApC/EBP and ApCREB2. ApAF is necessary for long-term facilitation induced by five pulses of serotonin, by activation of CREB1, or by derepression of ApCREB2. Overexpression of ApAF enhances the long-term facilitation further. Thus, ApAF is a candidate memory enhancer gene downstream from both CREB1 and ApCREB2.

A novel function for serotonin-mediated short-term facilitation in aplysia: conversion of a transient, cell-wide homosynaptic hebbian plasticity into a persistent, protein synthesis-independent synapse-specific enhancement.

Studies of sensitization and classical conditioning of the gill-withdrawal reflex in Aplysia have shown that the synaptic connections between identified glutamatergic sensory neurons and motor neurons can be enhanced in one of two ways: by a heterosynaptic (modulatory input-dependent) mechanism that gives rise with repetition to long-term facilitation and by a homosynaptic (activity-dependent) mechanism that gives rise with repetition to a facilitation that is partially blocked by 2-amino-5-phosphonovaleric acid and by injection of 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetate (BAPTA) into the postsynaptic cell and is similar to long-term potentiation in the hippocampus. We here have examined how these two forms of facilitation interact at the level of an individual synaptic connection by using a culture preparation consisting of a single bifurcated sensory neuron that forms independent synaptic contacts with each of two spatially separated motor neurons. We find that the homosynaptic facilitation produced by a train of action potentials is cell wide and is evident at all of the terminals of the sensory neuron. By contrast, the heterosynaptic facilitation mediated by the modulatory transmitter serotonin (5-HT) can operate at the level of a single synapse. Homosynaptic activation gives rise to only a transient facilitation lasting a few hours, even when repeated in a spaced manner. The heterosynaptic facilitation produced by a single pulse of 5-HT, applied to one terminal of the sensory neuron, also lasts only minutes. However, when one or more homosynaptic trains of spike activity are paired with even a single pulse of 5-HT applied to one of the two branches of the sensory neuron, the combined actions lead to a selective enhancement in synaptic strength only at the 5-HT-treated branch that now lasts more than a day, and thus amplifies, by more than 20-fold, the duration of the individually produced homo- and heterosynaptic facilitation. This form of synapse-specific facilitation has unusual long-term properties. It does not require protein synthesis, nor is it accompanied by synaptic growth.

Structural changes and the storage of long-term memory inAplysia

Long-term memory for sensitization of the gill-withdrawal reflex in Aplysia is associated with the growth of new synaptic connections between sensory and motor neurons. The duration of this structural change parallels the behavioral retention of the memory. Such changes can be reconstituted in dissociated cell culture by repeated presentations of the modulatory neurotransmitter serotonin (5HT) and are associated with an activity-dependent downregulation of NCAM-related cell adhesion molecules thought to contribute to cell recognition and axonal outgrowth during development. Thus, aspects of the mechanisms utilized for learning-related synaptic growth initiated by experience in the adult may eventually be understood in the context of the molecular logic that shapes synaptic circuitry during the later stages of neuronal development.

PKA-Dependent Regulation of Synaptic Enhancement between a Buccal Motor Neuron and Its Regulatory Interneuron in Lymnaea stagnalis

The cerebral giant cell (CGC) is known to play a crucial role in the regulation of feeding response in the pond snail, Lymnaea stagnalis. However, the mechanisms of signal transduction from the CGC to the following buccal motor neurons are not clear. In the present study, we examined whether cyclic AMP (cAMP)-dependent protein kinase (PKA) contributes to enhancement of a monosynaptic connection between the presynaptic CGC and the postsynaptic buccal motor neuron 1 (B1 cell). Injection of cAMP into the CGC or inhibition of phosphodiesterase by isobutylmethylxanthine in the CGC increased the amplitude of excitatory postsynaptic potential (EPSP) in the B1 cell, whereas no changes were detected in the electrical properties of the CGC. The synaptic enhancement in the B1 cell was completely blocked by inhibition of PKA in the CGC but did not require a de novo protein synthesis due to a PKA phosphorylation. The increase in the EPSP amplitude of B1 cell was associated with the increase in the amount of serotonin release from the CGC. These results hence provided the physiological evidence of the direct regulation of a synaptic enhancement by PKA in the CNS of L. stagnalis, indicating the completely different mechanism from that in the well-studied siphon- and gill-withdrawal reflex in Aplysia.

Classical conditioning, differential conditioning, and second-order conditioning of the Aplysia gill-withdrawal reflex in a simplified mantle organ preparation.

Nonassociative learning was previously examined in a simplified preparation consisting of the isolated mantle organs and abdominal ganglion of Aplysia californica that is advantageous for relating cellular events to behavior (T. E. Cohen, S. W. Kaplan, E. R. Kandel, & R. D. Hawkins, 1997). Results of the current study show that the gill-withdrawal reflex in that preparation also underwent 2 associative forms of learning: classical conditioning and differential conditioning. In addition, the reflex underwent second-order conditioning with either forward or simultaneous pairing of a novel conditioned stimulus (CS2) and a previously conditioned stimulus (CS1). Moreover, extinction of CS1 after simultaneous second-order conditioning was accompanied by a decrease in responding to CS2, suggesting that the conditioning might have involved formation of an association between the CSs. In each of these paradigms, learning in the Aplysia mantle organ preparation resembled learning in vertebrates.

Classical conditioning, differential conditioning, and second-order conditioning of the Aplysia gill-withdrawal reflex in a simplified mantle organ preparation.

Classical conditioning, differential conditioning, and second-order conditioning of the Aplysia gill-withdrawal reflex in a simplified mantle organ preparation.

A Simplified Preparation for Relating Cellular Events to Behavior: Mechanisms Contributing to Habituation, Dishabituation, and Sensitization of theAplysiaGill-Withdrawal Reflex

To relate cellular events to behavior in a more rigorous fashion, we have developed a simplified preparation for studying the gill-withdrawal reflex of Aplysia, in which it is relatively easy to record the activity of individual neurons during simple forms of learning. Approximately 84% of the reflex in this preparation is mediated through the single motor neuron LDG1, so that changes in the firing of LDG1 can account for most of the changes in behavior. We have used this preparation to investigate cellular mechanisms contributing to habituation, dishabituation, and sensitization by recording evoked firing, the complex postsynaptic potential (PSP), and the monosynaptic component of the complex PSP in LDG1. Our results suggest that habituation is largely attributable to depression at sensory neuron synapses. By contrast, dishabituation and sensitization involve several mechanisms at different loci, including facilitation at sensory neuron synapses, enhancement in the periphery (perhaps attributable to post-tetanic potentiation at the neuromuscular junction), and both facilitation and inhibition of excitatory and inhibitory interneurons. Moreover, these different mechanisms contribute preferentially at different times after training, so that information processing in the neuronal circuit for the reflex is distributed not only in space but also in time. Nonetheless, our results also suggest that the neuronal circuit is not a highly distributed neural network. Rather, plasticity of the reflex can evidently be accounted for by several specific mechanisms and loci of plasticity in a defined neural circuit, including a limited number of neurons, some of which make a large contribution to the behavior.

The dual effect of enflurane on gill withdrawal reflex ofAplysia

Superfusion of clinical concentrations of enflurane (0.5% or 1.0%), an inhalation anaesthetic, over the abdominal ganglion ofAplysia significantly affected the amplitude of the gill withdrawal reflex evoked by tactile stimulation of the siphon. Enflurane superfusion (0.5%) suppressed the gill withdrawal reflex amplitude (to 46.1% of control; P<0.001 vs control) in eight of ten experiments. In the remaining two experiments, enflurane superfusion of the abdominal ganglion significantly facilitated the gill withdrawal reflex amplitude (174.5% of control;P<0.01). In addition, enflurane superfusion significantly reduced the number of action potentials evoked in central gill motor neurons by the siphon stimulation (to 47.1% of control;P<0.01) in six out of nine experiments. In one of the remaining three experiments, enflurane increased the number of action potentials evoked by the stimulus (to 200.0% of control). In two of the three, enflurane did not alter the numbet of action potentials. Behavioural responses were ‘uncoupled’ from the neuronal responses as a result of enflurane superfusion.

The biochemistry of learning and memory.

An overview of some of the biochemical and molecular events involved in the process of learning and memory are presented in a short review. Two invertebrate models of learning are considered: the gill-withdrawal reflex of Aplysia and avoidance learning in Drosophila melanogaster. Particular attention is paid to the biochemical mechanisms underlying both the development of long-term potentiation (LTP) and passive avoidance learning (PAL) in the young chick. The role of several biological molecules in learning and memory are considered, for example, protein kinase C (PKC), Ca(++)-Calmodulin kinase II (CaMKII), GAP-43, and glutamate receptors.

A novel intermediate stage in the transition between short- and long-term facilitation in the sensory to motor neuron synapse of aplysia

A major difference between short- and long-term memory is that long-term memory is dependent on new protein synthesis. Long-term memory can be further subdivided into a transient, initial phase that is readily susceptible to disruption and a later, more stable and persistent stage. To analyze this transition on the cellular level, we have examined the steps whereby short-term facilitation is converted to a long-term form in the sensorimotor connection of the Aplysia gill-withdrawal reflex. We found that stable long-term facilitation (at 24 hr) requires a higher concentration (100 nM) of serotonin (5-HT) than does short-term facilitation (10 nM). By using low concentrations of 5-HT, which do not produce long-term facilitation, we now have been able to explore the intermediate phases between the short- and long-term processes. By this means we have uncovered a new transient phase that involves three mechanistically different mechanisms--covalent modification, translation, and transcription--each of which can be recruited as a function of the concentration of 5-HT.

Distributed aspects of the response to siphon touch in Aplysia: spread of stimulus information and cross-correlation analysis

We examined two aspects of the response to siphon stimulation in an attempt to test the hypothesis that the Aplysia CNS functions as a distributed system. First, we estimated the number of central neurons that respond to a light touch to the siphon skin. We made voltage-sensitive dye recordings from the abdominal, pleural, pedal, and cerebral ganglia. From these recordings we estimated that 220 abdominal neurons, 110 pleural neurons, and 650 pedal neurons were affected by the light touch. Thus, the information about this mild and localized stimulus is very widely distributed within the Aplysia CNS. This result allows the possibility that the Aplysia CNS functions as a distributed system. If only a small number of neurons had responded to the touch, it would have supported the conclusion that the gill-withdrawal reflex could be generated by a small, dedicated circuit. Second, we searched for correlations between the spike times of the individual abdominal ganglion neurons. Two time scales were examined: a millisecond time scale corresponding to the duration of a fast synaptic potential and a seconds time scale corresponding to the duration of the gill-withdrawal movement. Neuron pairs with highly correlated spike activity on a millisecond time scale must be connected by (or have a common input that uses) relatively powerful, fast, excitatory synapses. We expected that this kind of synaptic interaction would be relatively rare in nervous systems that functioned in a distributed manner. Indeed, only 0.3% of the neuron pairs had correlation coefficients of 0.15 or greater. These correlations accounted for approximately 2% of the action potentials generated in response to siphon stimulation. Thus, large, fast excitatory synaptic interactions appear to be relatively unimportant. This result is consistent with the hypothesis that the abdominal ganglion functions as a distributed system. When the longer time scale was used for the cross-correlograms, a large fraction of the cell pairs had correlated activity because many neurons are activated by the stimulus. It was not possible to interpret the slow correlations in terms of actual synaptic interactions between individual neurons. Our results are consistent with the possibility that the abdominal ganglion functions in a distributed manner. However, this evaluation is indirect and thus only tentative conclusions can be drawn. Evidence from several sources suggests that the neuronal interactions for generating the Aplysia gill-withdrawal reflex are complex.

C/EBP is an immediate-early gene required for the consolidation of long-term facilitation in Aplysia

The consolidation of long-term memory requires protein and mRNA synthesis. A similar requirement has been demonstrated for learning-related synaptic plasticity in the gill-withdrawal reflex of Aplysia. The monosynaptic component of this reflex can be reconstituted in vitro, where it undergoes both short- and long-term increases in synaptic strength in response to serotonin (5-HT), a neurotransmitter released during behavioral sensitization, a simple form of learning. As with sensitization, the long-term synaptic modification is characterized by a brief consolidation period during which gene expression is required. We find that during this phase, the transcription factor Aplysia CCAAT enhancer-binding protein (ApC/EBP) is induced rapidly by 5-HT and by cAMP, even in the presence of protein synthesis inhibitors. Blocking the function of ApC/EBP blocks long-term facilitation selectively without affecting the short-term process. These data indicate that cAMP-inducible immediate-early genes have an essential role in the consolidation of stable long-term synaptic plasticity in Aplysia.