Plasticity In Aplysia Research Articles

The CaV2\u03b11 EF-hand F helix tyrosine, a highly conserved locus for GPCR inhibition of CaV2 channels

The sensory neuron of Aplysia californica participates in several forms of presynaptic plasticity including homosynaptic depression, heterosynaptic depression, facilitation and the reversal of depression. The calcium channel triggering neurotransmitter release at most synapses is CaV2, consisting of the pore forming α1 subunit (CaV2α1), and auxiliary CaVβ, and CaVα2δ subunits. To determine the role of the CaV2 channel in presynaptic plasticity in Aplysia, we cloned Aplysia CaV2α1, CaVβ, and CaVα2δ and over-expressed the proteins in Aplysia sensory neurons (SN). We show expression of exogenous CaV2α1 in the neurites of cultured Aplysia SN. One proposed mechanism for heterosynaptic depression in Aplysia is through inhibition of CaV2. Here, we demonstrate that heterosynaptic depression of the CaV2 calcium current is inhibited when a channel with a Y-F mutation at the conserved Src phosphorylation site is expressed, showing the strong conservation of this mechanism over evolution. We also show that the Y-F mutation reduces heterosynaptic inhibition of neurotransmitter release, highlighting the physiological importance of this mechanism for the regulation of synaptic efficacy. These results also demonstrate our ability to replace endogenous CaV2 channels with recombinant channels allowing future examination of the structure function relationship of CaV2 in the regulation of transmitter release in this system.

MicroRNA-22 Gates Long-Term Heterosynaptic Plasticity in Aplysia through Presynaptic Regulation of CPEB and Downstream Targets.

The maintenance phase of memory-related long-term facilitation (LTF) of synapses between sensory and motor neurons of the gill-withdrawal reflex of Aplysia depends on a serotonin (5-HT)-triggered presynaptic upregulation of CPEB, a functional prion that regulates local protein synthesis at the synapse. The mechanisms whereby serotonin regulates CPEB levels in presynaptic sensory neurons are not known. Here, we describe a sensory neuron-specific microRNA 22 (miR-22) that has multiple binding sites on the mRNA of CPEB and inhibits it in the basal state. Serotonin triggers MAPK/Erk-dependent downregulation of miR-22, thereby upregulating the expression of CPEB, which in turn regulates, through functional CPE elements, the presynaptic expression of atypical PKC (aPKC), another candidate regulator of memory maintenance. Our findings support a model in which the neurotransmitter-triggered downregulation of miR-22 coordinates the regulation of genes contributing synergistically to the long-term maintenance of memory-related synaptic plasticity.

A novel fibroblast growth factor receptor family member promotes neuronal outgrowth and synaptic plasticity in aplysia.

Fibroblast Growth Factor (FGF) Receptors (FGFRs) regulate essential biological processes, including embryogenesis, angiogenesis, cellular growth and memory-related long-term synaptic plasticity. Whereas canonical FGFRs depend exclusively on extracellular Immunoglobulin (Ig)-like domains for ligand binding, other receptor types, including members of the tropomyosin-receptor-kinase (Trk) family, use either Ig-like or Leucine-Rich Repeat (LRR) motifs, or both. Little is known, however, about the evolutionary events leading to the differential incorporation of LRR domains into Ig-containing tyrosine kinase receptors. Moreover, although FGFRs have been identified in many vertebrate species, few reports describe their existence in invertebrates. Information about the biological relevance of invertebrate FGFRs and evolutionary divergences between them and their vertebrate counterparts is therefore limited. Here, we characterized ApLRRTK, a neuronal cell-surface protein recently identified in Aplysia. We unveiled ApLRRTK as the first member of the FGFRs family deprived of Ig-like domains that instead contains extracellular LRR domains. We describe that ApLRRTK exhibits properties typical of canonical vertebrate FGFRs, including promotion of FGF activity, enhancement of neuritic outgrowth and signaling via MAPK and the transcription factor CREB. ApLRRTK also enhanced the synaptic efficiency of neurons known to mediate in vivo memory-related defensive behaviors. These data reveal a novel molecular regulator of neuronal function in invertebrates, provide the first evolutionary linkage between LRR proteins and FGFRs and unveil an unprecedented mechanism of FGFR gene diversification in primeval central nervous systems.Electronic supplementary materialThe online version of this article (doi:10.1007/s00726-014-1803-2) contains supplementary material, which is available to authorized users.

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.

Characterization of prion-like conformational changes of the neuronal isoform of Aplysia CPEB

The neuronal isoform of cytoplasmic polyadenylation element-binding protein (CPEB) is a regulator of local protein synthesis at synapses and is critical in maintaining learning-related synaptic plasticity in Aplysia. Previous studies indicate that the function of Aplysia CPEB can be modulated by conversion to a stable prion-like state, thus contributing to the stabilization of long-term memory on a molecular level. Here, we used biophysical methods to demonstrate that Aplysia CPEB, like other prions, undergoes a conformational switch from soluble α-helix-rich oligomer to β-sheet-rich fiber in vitro. Solid-state NMR analyses of the fibers indicated a relatively rigid N-terminal prion domain. The fiber form of Aplysia CPEB showed enhanced binding to target mRNAs as compared to the soluble form. Consequently, we propose a model for the Aplysia CPEB fibers that may have relevance for functional prions in general.

State-dependent disruption of short-term facilitation due to overexpression of the apPDE4 supershort form in Aplysia.

Phosphodiesterases (PDEs) play important roles in synaptic plasticity by regulating cAMP signaling in various organisms. The supershort, short, and long forms of Aplysia PDE4 (apPDE4) have been cloned, and the long form has been shown to play a crucial role in 5- hydroxytryptamine (5-HT)-induced synaptic plasticity in Aplysia. To address the role of the supershort form in 5-HT-induced synaptic plasticity in Aplysia, we overexpressed the apPDE4 supershort form in Aplysia sensory neurons. Consequently, 5-HT-induced hyperexcitability and short-term facilitation in nondepressed synapses were blocked. However, the supershort form did not inhibit 5-HT-induced short-term facilitation in highly depressed synapses. These results show that the supershort form plays an important role in 5-HT-induced synaptic plasticity and disrupts it mainly by impairing cAMP signaling in Aplysia.

Neural mechanisms of operant conditioning and learning-induced behavioral plasticity in Aplysia.

Associative learning in goal-directed behaviors, in contrast to reflexive behaviors, can alter processes of decision-making in the selection of appropriate action and its initiation, thereby enabling animals, including humans, to gain a predictive understanding of their external environment. In the mollusc Aplysia, recent studies on appetitive operant conditioning in which the animal learns about the positive consequences of its behavior have provided insights into this form of associative learning which, although ubiquitous, remains mechanistically poorly understood. The findings support increasing evidence that central circuit- and cell-wide sites other than chemical synaptic connections, including electrical coupling and membrane conductances controlling intrinsic neuronal excitability and underlying voltage-dependent plateauing or oscillatory mechanisms, may serve as the neural substrates for behavioral plasticity resulting from operant conditioning. Aplysia therefore continues to provide a model system for understanding learning and memory formation that enables establishing the neurobiological links between behavioral, network, and cellular levels of analysis.

The Role of Rapid, Local, Postsynaptic Protein Synthesis in Learning-Related Synaptic Facilitation in Aplysia

The discovery that dendrites of neurons in the mammalian brain possess the capacity for protein synthesis stimulated interest in the potential role of local, postsynaptic protein synthesis in learning-related synaptic plasticity. But it remains unclear how local, postsynaptic protein synthesis actually mediates learning and memory in mammals. Accordingly, we examined whether learning in an invertebrate, the marine snail Aplysia, involves local, postsynaptic protein synthesis. Previously, we showed that the dishabituation and sensitization of the defensive withdrawal reflex in Aplysia require elevated postsynaptic Ca(2+), postsynaptic exocytosis, and functional upregulation of postsynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors. Here, we tested whether the synaptic facilitation that underlies dishabituation and sensitization in Aplysia requires local, postsynaptic protein synthesis. We found that the facilitatory transmitter, serotonin (5-HT), enhanced the response of the motor neuron to glutamate, the sensory neuron transmitter, and this enhancement depended on rapid protein synthesis. By using individual motor neurites surgically isolated from their cell bodies, we showed that the 5-HT-dependent protein synthesis occurred locally. Finally, by blocking postsynaptic protein synthesis, we disrupted the facilitation of the sensorimotor synapse. By demonstrating its critical role in a synaptic change that underlies learning and memory in a major model invertebrate system, our study suggests that local, postsynaptic protein synthesis is of fundamental importance to the cell biology of learning.

TGF-β1-Induced Long-Term Changes in Neuronal Excitability inAplysiaSensory Neurons Depend on MAPK

Transforming growth factor beta-1 (TGF-beta1) plays important roles in the early development of the nervous system and has been implicated in neuronal plasticity in adult organisms. It induces long-term increases in sensory neuron excitability in Aplysia as well as a long-term enhancement of synaptic efficacy at sensorimotor synapses. In addition, TGF-beta1 acutely regulates synapsin phosphorylation and reduces synaptic depression induced by low-frequency stimuli. Because of the critical role of MAPK in other forms of long-term plasticity in Aplysia, we examined the role of MAPK in TGF-beta1-induced long-term changes in neuronal excitability. Prolonged (6 h) exposure to TGF-beta1 induced long-term increases in excitability. We confirmed this finding and now report that exposure to TGF-beta1 was sufficient to activate MAPK and increase nuclear levels of active MAPK. Moreover, TGF-beta1 enhanced phosphorylation of the Aplysia transcriptional activator cAMP response element binding protein (CREB)1, a homologue to vertebrate CREB. Both the TGF-beta1-induced long-term changes in neuronal excitability and the phosphorylation of CREB1 were blocked in the presence of an inhibitor of the MAPK cascade, confirming a role for MAPK in long-term modulation of sensory neuron function.

Regulation of transcription factor C/ATF by the cAMP signal activation in hippocampal neurons, and molecular interaction of C/ATF with signal integrator CBP/p300

The CCAAT/enhancer binding proteins related activating transcription factor, C/ATF, is a mouse leucine-zipper transcription factor which is structurally homologous to ApCREB2, a suppressor integral to long-term synaptic plasticity in Aplysia. To gain a clue to whether C/ATF is involved in long-term plasticities of brain, we examined if the expression levels of C/ATF are modulated by cAMP, an inducer crucial for memory formation in Aplysia, Drosophila and mice. Our in situ hybridization analysis revealed the expression of C/ATF mRNA in hippocampal neurons. C/ATF protein levels increased after the cAMP signal stimulation in hippocampal neurons, while C/ATF mRNA levels remained constant. The human activating transcription factor 4 (hATF4), another homolog of ApCREB2, interacts with multiple domains of the coactivator CREB-binding protein (CBP), resulting in the potentiation of its ability to activate transcription. As expected, C/ATF was found to interact with three domains of CBP including CREB binding domain or kinase-inducible interaction (KIX) domain, the third cysteine–histidine-rich region (CH3 domain) and the nuclear receptor coactivator p160/SRC-1-interacting domain. Interestingly, C/ATF was further found to interact strongly with CREB binding protein/p300 (CBP/p300) CH1 domain. Mammalian two hybrid assays indicated that the interaction between C/ATF and CBP/p300 can occur in mammalian cells, and that the p300 CH1 domain is critical for the interaction. Thus, C/ATF may be implicated in transcription-dependent phase of hippocampal long-term plasticities through the modulation of its protein level under cAMP signal and the interaction with signal integrator, CBP/p300.

TGF-β1 inAplysia: Role in Long-Term Changes in the Excitability of Sensory Neurons and Distribution of TβR-II-Like Immunoreactivity

Exogenous recombinant human transforming growth factor beta-1 (TGF-beta1) induced long-term facilitation of Aplysia sensory-motor synapses. In addition, 5-HT-induced facilitation was blocked by application of a soluble fragment of the extracellular portion of the TGF-beta1 type II receptor (TbetaR-II), which presumably acted by scavenging an endogenous TGF-beta1-like molecule. Because TbetaR-II is essential for transmembrane signaling by TGF-beta, we sought to determine whether Aplysia tissues contained TbetaR-II and specifically, whether neurons expressed the receptor. Western blot analysis of Aplysia tissue extracts demonstrated the presence of a TbetaR-II-immunoreactive protein in several tissue types. The expression and distribution of TbetaR-II-immunoreactive proteins in the central nervous system was examined by immunohistochemistry to elucidate sites that may be responsive to TGF-beta1 and thus may play a role in synaptic plasticity. Sensory neurons in the ventral-caudal cluster of the pleural ganglion were immunoreactive for TbetaR-II, as well as many neurons in the pedal, abdominal, buccal, and cerebral ganglia. Sensory neurons cultured in isolation and cocultured sensory and motor neurons were also immunoreactive. TGF-beta1 affected the biophysical properties of cultured sensory neurons, inducing an increase of excitability that persisted for at least 48 hr. Furthermore, exposure to TGF-beta1 resulted in a reduction in the firing threshold of sensory neurons. These results provide further support for the hypothesis that TGF-beta1 plays a role in long-term synaptic plasticity in Aplysia.

A genetic switch for long-term memory

Current models of brain function hold that learning corresponds to changes in the efficacy of single synapses. The study of learning and of a variety of forms of synaptic plasticity has revealed that both have at least two phases: an early phase that is not dependent on protein synthesis and a late phase that depends on new transcription and translation. Our laboratory has examined synaptic plasticity in Aplysia and in mice to better understand the regulatory events that lead to the induction of the late, protein synthesis-dependent phase of synaptic plasticity. Our recent studies of Aplysia have revealed that the genes that control the late phase of synaptic facilitation are controlled by both an activator, ApCREB1, and a repressor, ApCREB2. This leads to a model in which the late phase of synaptic facilitation is initiated by a perturbation of the balance between activators and repressors of transcription; this perturbation can be accomplished by regulating the activator, the repressor, or both. We, and others, have shown that this transcriptional switch is conserved, at least in part, in the regulation of synaptic plasticity in mice: CREB is inplicated in activation of genes required for LTP, a model for synaptic plasticity in the mammalian hippocampus. We speculate that a similar balance between activators and repressors may regulate the genes required for long-term memory in mammals.

CAMP modulates an S-type K+ channel coupled to GABAB receptors in mammalian respiratory neurons.

Premotor respiratory neurons from neonatal rats express a Ba(2+)-insensitive outward rectifying K+ channel (KOR) which is activated by gamma-aminobutyric acid acting at its beta receptor. The biophysical properties of KOR are similar to those described for the S-channel (KS) which underlies simple forms of non-associative learning in the marine mollusc Aplysia. We show here that KOR, like the S-channel, is inhibited by cAMP. In addition, we demonstrate that this inhibition is due to a change in closed time kinetics. Our data suggests that the ionic and biochemical substrates underlying synaptic plasticity in Aplysia have been phylogenetically conserved in mammalian motor circuits such as that controlling rhythmic breathing movements.

Effects on protein synthesis produced by pairing depolarization with serotonin, an analogue of associative learning in Aplysia.

A form of associative plasticity in Aplysia, activity-dependent neuromodulation, involves the convergence of neuronal activity and the effects of a modulatory transmitter. To investigate the role of protein synthesis in associative plasticity, we examined the effects of a biochemical analogue of activity-dependent neuromodulation on the level of incorporation of labeled amino acid into proteins. To mimic associative training, abdominal ganglia were exposed to paired treatments of a depolarizing agent, elevated potassium, and a modulatory transmitter, serotonin. The effects of elevated potassium and serotonin applied alone were also examined. At least two proteins (nos. 9 and 17) were affected in a nonadditive way by the paired procedure. Incorporation of label into protein 9 was increased by the paired procedure but was not affected by either elevated potassium or serotonin. Incorporation of label into protein 17 was significantly affected by elevated potassium or serotonin, but the effect of the paired procedure was significantly less than the summed effects of elevated potassium and serotonin applied alone. These results indicate that changes in protein synthesis may be important in the induction of associative plasticities. Amino acid sequences of two peptides derived from protein 9 were obtained. Then, a partial cDNA clone for protein 9 was obtained by performing PCR with degenerate primers corresponding to portions of the sequences of the two peptides. The sequence of protein 9 is related to sequences previously reported for a family of genes comprising the stringent starvation protein of Escherichia coli, auxin-induced proteins of plants, and glutathione S-transferases of a number of organisms.

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.

The role of interneurons in controlling the tail-withdrawal reflex in Aplysia: a network model

1. The contributions of monosynaptic and polysynaptic circuitry to the tail-withdrawal reflex in the marine mollusk Aplysia californica were assessed by the use of physiologically based neural network models. Effects of monosynaptic circuitry were examined by the use of a two-layer network model with four sensory neurons in the input layer and one motor neuron in the output layer. Results of these simulations indicated that the monosynaptic circuit could not account fully for long-duration responses of tail motor neurons elicited by tail stimulation. 2. A three-layer network model was constructed by interposing a layer of two excitatory interneurons between the input and output layers of the two-layer network model. These interneurons had properties mimicking those of the recently described interneuron LP117, receiving excitatory input from pleural sensory neurons and evoking a biphasic excitatory postsynaptic potential (EPSP) in pedal motor neurons (Cleary and Byrne 1993). The three-layer model could account for long-duration responses in motor neurons. 3. Sensory neurons are a known site of plasticity in Aplysia. Synaptic plasticity was incorporated into the three-layer model by altering the magnitudes of conductance changes evoked in motor neurons and interneurons by presynaptic sensory neurons. In these simulations the excitatory interneurons converted an amplitude-coded input into an amplitude- and duration-coded output, allowing the three-layer network to support a large range of output amplitudes and durations. 4. Synaptic plasticity at more than one locus modified dramatically the input-output relationship of the three-layer network model. This feature gave the model redundancy in its plastic properties and points to the possibility of distributed memory in the circuitry mediating withdrawal reflexes in Aplysia. Multiple sites of control over the response of the network would likely allow a more diverse repertoire of responses.

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.

Inhibitors of protein and RNA synthesis block structural changes that accompany long-term heterosynaptic plasticity in Aplysia

Synaptic connections between the sensory and motor neurons of Aplysia in culture undergo long-term facilitation in response to serotonin (5-HT) and long-term depression in response to FMRFamide. These long-term functional changes are dependent on the synthesis of macromolecules during the period in which the transmitter is applied and are accompanied by structural changes. There is an increase and a decrease, respectively, in the number of sensory neuron varicosities in response to 5-HT and FMRFamide. To determine whether macromolecular synthesis is also required for the structural changes, we examined in parallel the effects of inhibitors of protein (anisomycin) or RNA (actinomycin D) synthesis on the structural and functional changes. We have found that anisomycin and actinomycin D block both the enduring alterations in varicosity number and the long-lasting changes in synaptic potential. These results indicate that macromolecular synthesis is required for expression of the long-lasting structural changes in the sensory cells and that this synthesis is correlated with the long-term functional modulation of sensorimotor synapses.

A Functional, Cellular, and Evolutionary Model of Nociceptive Plasticity in Aplysia.

Nociceptive plasticity is defined as behavioral and cellular modification produced by activation of nociceptors. A brief survey of nociceptive plasticity in Aplysia reveals a puzzling mixture of behavioral modifications of opposite sign and widely varying durations. These include general sensitization, site-specific sensitization, response-specific facilitation, and inhibition of defensive responses. This behavioral complexity is more than matched by the complexity of cellular correlates reported for the behavioral modifications. A functional model is proposed linking complex patterns of behavioral and neural plasticity in Aplysia to potentially general principles of nociceptive function. This model is centered around three overlapping but functionally distinct phases: injury detection, escape, and recuperation. A hypothesis about the early origin of nociceptive plasticity in primitive mechanosensory neurons is then developed, based on similarities in the organization and modifiability of nociceptive systems in evolutionarily divergent groups (primarily mollusks and mammals) and on inferences about the early adaptiveness of postinjury behavioral plasticity. Preliminary evidence suggests that aspects of nociceptive plasticity, and perhaps other forms of memory, may have been derived from cellular repair and signal compensation mechanisms.