RESUMO
Small RNA-mediated gene regulation during development causes long-lasting changes in cellular phenotypes. To determine whether small RNAs of the adult brain can regulate memory storage, a process that requires stable and long-lasting changes in the functional state of neurons, we generated small RNA libraries from the Aplysia CNS. In these libraries, we discovered an unexpectedly abundant expression of a 28 nucleotide sized class of piRNAs in brain, which had been thought to be germline specific. These piRNAs have unique biogenesis patterns, predominant nuclear localization, and robust sensitivity to serotonin, a modulatory transmitter that is important for memory. We find that the Piwi/piRNA complex facilitates serotonin-dependent methylation of a conserved CpG island in the promoter of CREB2, the major inhibitory constraint of memory in Aplysia, leading to enhanced long-term synaptic facilitation. These findings provide a small RNA-mediated gene regulatory mechanism for establishing stable long-term changes in neurons for the persistence of memory.
Assuntos
Epigenômica , Memória , Plasticidade Neuronal , Neurônios/fisiologia , RNA Interferente Pequeno/metabolismo , Animais , Aplysia/metabolismo , Sequência de Bases , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Sinapses/metabolismoRESUMO
A long-standing question in the study of long-term memory is how a memory trace persists for years when the proteins that initiated the process turn over and disappear within days. Previously, we postulated that self-sustaining amyloidogenic oligomers of cytoplasmic polyadenylation element-binding protein (CPEB) provide a mechanism for the maintenance of activity-dependent synaptic changes and, thus, the persistence of memory. Here, we found that the Drosophila CPEB Orb2 forms amyloid-like oligomers, and oligomers are enriched in the synaptic membrane fraction. Of the two protein isoforms of Orb2, the amyloid-like oligomer formation is dependent on the Orb2A form. A point mutation in the prion-like domain of Orb2A, which reduced amyloid-like oligomerization of Orb2, did not interfere with learning or memory persisting up to 24 hr. However the mutant flies failed to stabilize memory beyond 48 hr. These results support the idea that amyloid-like oligomers of neuronal CPEB are critical for the persistence of long-term memory.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/fisiologia , Fatores de Transcrição/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Sequência de Aminoácidos , Amiloide/metabolismo , Animais , Aplysia/metabolismo , Encéfalo/metabolismo , Proteínas de Drosophila/química , Memória , Dados de Sequência Molecular , Neurônios/metabolismo , Mutação Puntual , Isoformas de Proteínas/metabolismo , Sinapses/metabolismo , Fatores de Transcrição/química , Fatores de Poliadenilação e Clivagem de mRNA/químicaRESUMO
The l- to d-amino acid residue isomerization of neuropeptides is an understudied post-translational modification found in animals across several phyla. Despite its physiological importance, little information is available regarding the impact of endogenous peptide isomerization on receptor recognition and activation. As a result, the full roles peptide isomerization play in biology are not well understood. Here, we identify that the Aplysia allatotropin-related peptide (ATRP) signaling system utilizes l- to d-residue isomerization of one amino acid residue in the neuropeptide ligand to modulate selectivity between two distinct G protein-coupled receptors (GPCRs). We first identified a novel receptor for ATRP that is selective for the D2-ATRP form, which bears a single d-phenylalanine residue at position 2. Using cell-based receptor activation experiments, we then characterized the stereoselectivity of the two known ATRP receptors for both endogenous ATRP diastereomers, as well as for homologous toxin peptides from a carnivorous predator. We found that the ATRP system displayed dual signaling through both the Gαq and Gαs pathways, and each receptor was selectively activated by one naturally occurring ligand diastereomer over the other. Overall, our results provide insights into an unexplored mechanism by which nature regulates intercellular communication. Given the challenges in detecting l- to d-residue isomerization from complex mixtures de novo and in identifying receptors for novel neuropeptides, it is likely that other neuropeptide-receptor systems may also utilize changes in stereochemistry to modulate receptor selectivity in a manner similar to that discovered here.
Assuntos
Aminoácidos , Receptores de Neuropeptídeos , Animais , Isomerismo , Ligantes , Fenilalanina , AplysiaRESUMO
Transforming growth factor ß (TGFß) is required for long-term memory (LTM) for sensitization in Aplysia. When LTM is induced using a two-trial training protocol, TGFß inhibition only blocks LTM when administrated at the second, not the first trial. Here, we show that TGFß acts as a "repetition detector" during the induction of two-trial LTM. Secretion of the biologically inert TGFß proligand must coincide with its proteolytic activation by the Bone morphogenetic protein-1 (BMP-1/Tolloid) metalloprotease, which occurs specifically during trial two of our two-trial training paradigm. This paradigm establishes long-term synaptic facilitation (LTF), the cellular correlate of LTM. BMP-1 application paired with a single serotonin (5HT) pulse induced LTF, whereas neither a single 5HT pulse nor BMP-1 alone effectively did so. On the other hand, inhibition of endogenous BMP-1 activity blocked the induction of two-trial LTF. These results suggest a unique role for TGFß in the interaction of repeated trials: during learning, repeated stimuli engage separate steps of the TGFß cascade that together are necessary for the induction of long-lasting memories.
Assuntos
Potenciação de Longa Duração , Fator de Crescimento Transformador beta , Animais , Potenciação de Longa Duração/fisiologia , Fator de Crescimento Transformador beta/farmacologia , Plasticidade Neuronal/fisiologia , Memória de Longo Prazo/fisiologia , Aplysia/fisiologiaRESUMO
The functional switch of glutamine/asparagine (Q/N)-rich prions and the neurotoxicity of polyQ-expanded proteins involve complex aggregation-prone structural transitions, commonly presumed to be forming ß sheets. By analyzing sequences of interaction partners of these proteins, we discovered a recurrent presence of coiled-coil domains both in the partners and in segments that flank or overlap Q/N-rich and polyQ domains. Since coiled coils can mediate protein interactions and multimerization, we studied their possible involvement in Q/N-rich and polyQ aggregations. Using circular dichroism and chemical crosslinking, we found that Q/N-rich and polyQ peptides form α-helical coiled coils in vitro and assemble into multimers. Using structure-guided mutagenesis, we found that coiled-coil domains modulate in vivo properties of two Q/N-rich prions and polyQ-expanded huntingtin. Mutations that disrupt coiled coils impair aggregation and activity, whereas mutations that enhance coiled-coil propensity promote aggregation. These findings support a coiled-coil model for the functional switch of Q/N-rich prions and for the pathogenesis of polyQ-expansion diseases.
Assuntos
Aplysia/metabolismo , Príons/química , Príons/metabolismo , Sequência de Aminoácidos , Animais , Aplysia/química , Dicroísmo Circular , Glutationa Peroxidase/metabolismo , Proteína Huntingtina , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Peptídeos/metabolismo , Príons/genética , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Prions are proteins that can assume at least two distinct conformational states, one of which is dominant and self-perpetuating. Previously we found that a translation regulator CPEB from Aplysia, ApCPEB, that stabilizes activity-dependent changes in synaptic efficacy can display prion-like properties in yeast. Here we find that, when exogenously expressed in sensory neurons, ApCPEB can form an amyloidogenic self-sustaining multimer, consistent with it being a prion-like protein. In addition, we find that conversion of both the exogenous and the endogenous ApCPEB to the multimeric state is enhanced by the neurotransmitter serotonin and that an antibody that recognizes preferentially the multimeric ApCPEB blocks persistence of synaptic facilitation. These results are consistent with the idea that ApCPEB can act as a self-sustaining prion-like protein in the nervous system and thereby might allow the activity-dependent change in synaptic efficacy to persist for long periods of time.
Assuntos
Aplysia/metabolismo , Príons/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Amiloide/metabolismo , Animais , Potenciação de Longa Duração , Polilisina/metabolismo , Príons/química , Células Receptoras Sensoriais/metabolismo , Serotonina/metabolismo , Sinapses/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/químicaRESUMO
The gastropod mollusk Aplysia is an important model for cellular and molecular neurobiological studies, particularly for investigations of molecular mechanisms of learning and memory. We developed an optimized assembly pipeline to generate an improved Aplysia nervous system transcriptome. This improved transcriptome enabled us to explore the evolution of cognitive capacity at the molecular level. Were there evolutionary expansions of neuronal genes between this relatively simple gastropod Aplysia (20,000 neurons) and Octopus (500 million neurons), the invertebrate with the most elaborate neuronal circuitry and greatest behavioral complexity? Are the tremendous advances in cognitive power in vertebrates explained by expansion of the synaptic proteome that resulted from multiple rounds of whole genome duplication in this clade? Overall, the complement of genes linked to neuronal function is similar between Octopus and Aplysia. As expected, a number of synaptic scaffold proteins have more isoforms in humans than in Aplysia or Octopus. However, several scaffold families present in mollusks and other protostomes are absent in vertebrates, including the Fifes, Lev10s, SOLs, and a NETO family. Thus, whereas vertebrates have more scaffold isoforms from select families, invertebrates have additional scaffold protein families not found in vertebrates. This analysis provides insights into the evolution of the synaptic proteome. Both synaptic proteins and synaptic plasticity evolved gradually, yet the last deuterostome-protostome common ancestor already possessed an elaborate suite of genes associated with synaptic function, and critical for synaptic plasticity.
Assuntos
Aplysia , Evolução Biológica , Cognição , Sinapses , Animais , Aplysia/genética , Aplysia/metabolismo , Plasticidade Neuronal/genética , Neurônios/metabolismo , Isoformas de Proteínas/genética , Proteoma , Sinapses/metabolismo , TranscriptomaRESUMO
Two-trial learning in Aplysia reveals nonlinear interactions between training trials: A single trial has no effect, but two precisely spaced trials induce long-term memory. Extracellularly regulated kinase (ERK) activity is essential for intertrial interactions, but the mechanism remains unresolved. A combination of immunochemical and optogenetic tools reveals unexpected complexity of ERK signaling during the induction of long-term synaptic facilitation by two spaced pulses of serotonin (5-hydroxytryptamine, 5HT). Specifically, dual ERK phosphorylation at its activating TxY motif is accompanied by dephosphorylation at the pT position, leading to a buildup of inactive, singly phosphorylated pY-ERK. Phosphorylation and dephosphorylation occur concurrently but scale differently with varying 5HT concentrations, predicting that mixed two-trial protocols involving both "strong" and "weak" 5HT pulses should be sensitive to the precise order and timing of trials. Indeed, long-term synaptic facilitation is induced only when weak pulses precede strong, not vice versa. This may represent a physiological mechanism to prioritize memory of escalating threats.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular , Memória de Longo Prazo , Priming de Repetição , Serotonina , Animais , Aplysia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Memória de Longo Prazo/fisiologia , Optogenética , Fosforilação/genética , Priming de Repetição/fisiologia , Serotonina/farmacologia , Fatores de TempoRESUMO
Changes caused by learning that a food is inedible in Aplysia were examined for fast and slow synaptic connections from the buccal ganglia S1 cluster of mechanoafferents to five followers, in response to repeated stimulus trains. Learning affected only fast connections. For these, unique patterns of change were present in each follower, indicating that learning differentially affects the different branches of the mechanoafferents to their followers. In some followers, there were increases in either excitatory or inhibitory connections, and in others, there were decreases. Changes in connectivity resulted from changes in the amplitude of excitation or inhibition, or as a result of the number of connections, or of both. Some followers also exhibited changes in either within or between stimulus train plasticity as a result of learning. In one follower, changes differed from the different areas of the S1 cluster. The patterns of changes in connectivity were consistent with the behavioral changes produced by learning, in that they would produce an increase in the bias to reject or to release food, and a decrease in the likelihood to respond to food.
Assuntos
Aplysia , Gânglios dos Invertebrados , Neurônios Motores , Aplysia/fisiologia , Animais , Neurônios Motores/fisiologia , Gânglios dos Invertebrados/fisiologia , Aprendizagem/fisiologia , Mecanorreceptores/fisiologia , Plasticidade Neuronal/fisiologia , Alimentos , Comportamento Alimentar/fisiologiaRESUMO
How does repeated stimulation of mechanoafferents affect feeding motor neurons? Monosynaptic connections from a mechanoafferent population in the Aplysia buccal ganglia to five motor followers with different functions were examined during repeated stimulus trains. The mechanoafferents produced both fast and slow synaptic outputs, which could be excitatory or inhibitory. In contrast, other Aplysia mechanoafferents produce only fast excitation on their followers. In addition, patterns of synaptic connections were different to the different motor followers. Some followers received both fast excitation and fast inhibition, whereas others received exclusively fast excitation. All followers showed strong decreases in fast postsynaptic potential (PSP) amplitude within a stimulus train. Fast and slow synaptic connections were of net opposite signs in some followers but not in others. For one follower, synaptic contacts were not uniform from all subareas of the mechanoafferent cluster. Differences in properties of the buccal ganglia mechanoafferents and other Aplysia mechanoafferents may arise because the buccal ganglia neurons innervate the interior of the feeding apparatus, rather than an external surface, and connect to motor neurons for muscles with different motor functions. Fast connection patterns suggest that these synapses may be activated when food slips, biasing the musculature to release food. The largest slow inhibitory synaptic PSPs may contribute to a delay in the onset of the next behavior. Additional functions are also possible.
Assuntos
Aplysia , Comportamento Alimentar , Gânglios dos Invertebrados , Neurônios Motores , Animais , Aplysia/fisiologia , Neurônios Motores/fisiologia , Gânglios dos Invertebrados/fisiologia , Comportamento Alimentar/fisiologia , Mecanorreceptores/fisiologia , Sinapses/fisiologia , Estimulação FísicaRESUMO
Peptidergic dense-core vesicles are involved in packaging and releasing neuropeptides and peptide hormones-critical processes underlying brain, endocrine and exocrine function. Yet, the heterogeneity within these organelles, even for morphologically defined vesicle types, is not well characterized because of their small volumes. We present image-guided, high-throughput mass spectrometry-based protocols to chemically profile large populations of both dense-core vesicles and lucent vesicles for their lipid and peptide contents, allowing observation of the chemical heterogeneity within and between these two vesicle populations. The proteolytic processing products of four prohormones are observed within the dense-core vesicles, and the mass spectral features corresponding to the specific peptide products suggest three distinct dense-core vesicle populations. Notable differences in the lipid mass range are observed between the dense-core and lucent vesicles. These single-organelle mass spectrometry approaches are adaptable to characterize a range of subcellular structures.
Assuntos
Aplysia/citologia , Ensaios de Triagem em Larga Escala/métodos , Aprendizado de Máquina , Organelas/fisiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , AnimaisRESUMO
The mechanical forces experienced during movement and the time constants of muscle activation are important determinants of the durations of behaviours, which may both be affected by size-dependent scaling. The mechanics of slow movements in small animals are dominated by elastic forces and are thus quasistatic (i.e. always near mechanical equilibrium). Muscular forces producing movement and elastic forces resisting movement should scale identically (proportional to mass2/3), leaving the scaling of the time constant of muscle activation to play a critical role in determining behavioural duration. We tested this hypothesis by measuring the duration of feeding behaviours in the marine mollusc Aplysia californica whose body sizes spanned three orders of magnitude. The duration of muscle activation was determined by measuring the time it took for muscles to produce maximum force as A. californica attempted to feed on tethered inedible seaweed, which provided an in vivo approximation of an isometric contraction. The timing of muscle activation scaled with mass0.3. The total duration of biting behaviours scaled identically, with mass0.3, indicating a lack of additional mechanical effects. The duration of swallowing behaviour, however, exhibited a shallower scaling of mass0.17. We suggest that this was due to the allometric growth of the anterior retractor muscle during development, as measured by micro-computed tomography (micro-CT) scans of buccal masses. Consequently, larger A. californica did not need to activate their muscles as fully to produce equivalent forces. These results indicate that muscle activation may be an important determinant of the scaling of behavioural durations in quasistatic systems.
Assuntos
Aplysia , Músculos , Animais , Aplysia/fisiologia , Microtomografia por Raio-X , Músculos/fisiologia , Comportamento Alimentar/fisiologia , Deglutição/fisiologiaRESUMO
The coordination of complex behavior requires knowledge of both neural dynamics and the mechanics of the periphery. The feeding system of Aplysia californica is an excellent model for investigating questions in soft body systems' neuromechanics because of its experimental tractability. Prior work has attempted to elucidate the mechanical properties of the periphery by using a Hill-type muscle model to characterize the force generation capabilities of the key protractor muscle responsible for moving Aplysia's grasper anteriorly, the I2 muscle. However, the I1/I3 muscle, which is the main driver of retractions of Aplysia's grasper, has not been characterized. Because of the importance of the musculature's properties in generating functional behavior, understanding the properties of muscles like the I1/I3 complex may help to create more realistic simulations of the feeding behavior of Aplysia, which can aid in greater understanding of the neuromechanics of soft-bodied systems. To bridge this gap, in this work, the I1/I3 muscle complex was characterized using force-frequency, length-tension, and force-velocity experiments and showed that a Hill-type model can accurately predict its force-generation properties. Furthermore, the muscle's peak isometric force and stiffness were found to exceed those of the I2 muscle, and these results were analyzed in the context of prior studies on the I1/I3 complex's kinematics in vivo.
Assuntos
Aplysia , Aplysia/fisiologia , Animais , Fenômenos Biomecânicos/fisiologia , Modelos Biológicos , Comportamento Alimentar/fisiologia , Contração Muscular/fisiologia , Músculos/fisiologia , Músculo Esquelético/fisiologiaRESUMO
Studying the nervous system underlying animal motor control can shed light on how animals can adapt flexibly to a changing environment. We focus on the neural basis of feeding control in Aplysia californica. Using the Synthetic Nervous System framework, we developed a model of Aplysia feeding neural circuitry that balances neurophysiological plausibility and computational complexity. The circuitry includes neurons, synapses, and feedback pathways identified in existing literature. We organized the neurons into three layers and five subnetworks according to their functional roles. Simulation results demonstrate that the circuitry model can capture the intrinsic dynamics at neuronal and network levels. When combined with a simplified peripheral biomechanical model, it is sufficient to mediate three animal-like feeding behaviors (biting, swallowing, and rejection). The kinematic, dynamic, and neural responses of the model also share similar features with animal data. These results emphasize the functional roles of sensory feedback during feeding.
Assuntos
Aplysia , Retroalimentação Sensorial , Comportamento Alimentar , Modelos Neurológicos , Animais , Aplysia/fisiologia , Comportamento Alimentar/fisiologia , Retroalimentação Sensorial/fisiologia , Simulação por Computador , Neurônios/fisiologia , Rede Nervosa/fisiologia , Fenômenos Biomecânicos , Redes Neurais de ComputaçãoRESUMO
Gonadotropin-releasing hormone (GnRH) superfamily comprises multiple families of signaling peptides in both protostomes and deuterostomes. Among this superfamily, vertebrate GnRH stimulates reproduction, but other GnRH superfamily members elicit diverse pleiotropic effects. Within the GnRH superfamily members, adipokinetic hormone (AKH) and its receptor are well described in ecdysozoans but understudied in other lineages. To fill this knowledge gap, we deorphanized a putative receptor for a lophotrochozoan AKH in a gastropod mollusk, Aplysia californica, and named it Aplca-AKHR. Phylogenetic analysis revealed an orthologous relationship of Aplca-AKHR with ecdysozoan AKHRs and other putative lophotrochozoan AKHRs. Aplca-AKHR bound specifically to the previously identified Aplca-AKH with high affinity and activated the inositol phosphate pathway. Aplca-AKHR was expressed widely among central and peripheral tissues, but most prominently in several central ganglia and the heart. The expression of Aplca-AKHR was downregulated by a hyposaline challenge, consistent with a role in volume and fluid regulation previously described for its ligand, Aplca-AKH. In summary, this is the first pairing of a lophotrochozoan AKH with its cognate receptor. Expression data further support diverse central and peripheral roles, including volume and fluid control, of this ligand/receptor pair.
Assuntos
Gastrópodes , Hormônios de Inseto , Animais , Aplysia/genética , Aplysia/metabolismo , Sequência de Aminoácidos , Gastrópodes/metabolismo , Filogenia , Ligantes , Hormônio Liberador de Gonadotropina/metabolismo , Hormônios de Inseto/metabolismo , Ácido Pirrolidonocarboxílico/metabolismoRESUMO
Habituation and sensitization (nonassociative learning) are among the most fundamental forms of learning and memory behavior present in organisms that enable adaptation and learning in dynamic environments. Emulating such features of intelligence found in nature in the solid state can serve as inspiration for algorithmic simulations in artificial neural networks and potential use in neuromorphic computing. Here, we demonstrate nonassociative learning with a prototypical Mott insulator, nickel oxide (NiO), under a variety of external stimuli at and above room temperature. Similar to biological species such as Aplysia, habituation and sensitization of NiO possess time-dependent plasticity relying on both strength and time interval between stimuli. A combination of experimental approaches and first-principles calculations reveals that such learning behavior of NiO results from dynamic modulation of its defect and electronic structure. An artificial neural network model inspired by such nonassociative learning is simulated to show advantages for an unsupervised clustering task in accuracy and reducing catastrophic interference, which could help mitigate the stability-plasticity dilemma. Mott insulators can therefore serve as building blocks to examine learning behavior noted in biology and inspire new learning algorithms for artificial intelligence.
Assuntos
Algoritmos , Aplysia/fisiologia , Inteligência Artificial , Elementos Isolantes , Redes Neurais de Computação , Níquel/química , Sinapses/fisiologia , Animais , Elétrons , Modelos Neurológicos , Plasticidade NeuronalRESUMO
An in vitro analog of learning that a food is inedible provided insight into mechanisms underlying the learning. Aplysia learn to stop responding to a food when they attempt but fail to swallow it. Pairing a cholinergic agonist with an NO donor or histamine in the Aplysia cerebral ganglion produced significant decreases in fictive feeding in response to the cholinergic agonist alone. Acetylcholine (ACh) is the transmitter of chemoreceptors sensing food touching the lips. Nitric oxide (NO) and histamine (HA) signal failed attempts to swallow food. Reduced responses to the cholinergic agonist after pairing with NO or HA indicate that learning partially arises via a decreased response to ACh in the cerebral ganglion.
Assuntos
Aplysia , Deglutição , Animais , Aplysia/fisiologia , Deglutição/fisiologia , Histamina , Comportamento Alimentar/fisiologia , Óxido Nítrico/fisiologia , Agonistas ColinérgicosRESUMO
Neuropeptides are widely used as neurotransmitters in vertebrates and invertebrates. In vertebrates, a detailed understanding of their functions as transmitters has been hampered by the complexity of the nervous system. The marine mollusk Aplysia, with a simpler nervous system and many large, identified neurons, presents several advantages for addressing this question and has been used to examine the roles of tens of peptides in behavior. To screen for other peptides that might also play roles in behavior, we observed immunoreactivity in individual neurons in the central nervous system of adult Aplysia with antisera raised against the Aplysia peptide FMRFamide and two mammalian peptides that are also found in Aplysia, cholecystokinin (CCK) and neuropeptide Y (NPY), as well as serotonin (5HT). In addition, we observed staining of individual neurons with antisera raised against mammalian somatostatin (SOM) and peptide histidine isoleucine (PHI). However, genomic analysis has shown that these two peptides are not expressed in the Aplysia nervous system, and we have therefore labeled the unknown peptides stained by these two antibodies as XSOM and XPHI There was an area at the anterior end of the cerebral ganglion that had staining by antisera raised against many different transmitters, suggesting that this may be a modulatory region of the nervous system. There was also staining for XSOM and, in some cases, FMRFamide in the bag cell cluster of the abdominal ganglion. In addition, these and other studies have revealed a fairly high degree of colocalization of different neuropeptides in individual neurons, suggesting that the peptides do not just act independently but can also interact in different combinations to produce complex functions. The simple nervous system of Aplysia is advantageous for further testing these ideas.
Assuntos
Aplysia , Neuropeptídeos , Animais , Aplysia/fisiologia , FMRFamida , Sistema Nervoso Central/química , Gânglios/química , MamíferosRESUMO
Despite numerous studies examining the mechanisms of operant conditioning (OC), the diversity of OC plasticity loci and their synergism have not been examined sufficiently. In the well-characterized feeding neural circuit of Aplysia, in vivo and in vitro appetitive OC increases neuronal excitability and electrical coupling among several neurons leading to an increase in expression of ingestive behavior. Here, we used the in vitro analog of OC to investigate whether OC reduces the excitability of a neuron, B4, whose inhibitory connections decrease expression of ingestive behavior. We found OC decreased the excitability of B4. This change appeared intrinsic to B4 because it could be replicated with an analog of OC in isolated cultures of B4 neurons. In addition to changes in B4 excitability, OC decreased the strength of B4's inhibitory connection to a key decision-making neuron, B51. The OC-induced changes were specific without affecting the excitability of another neuron critical for feeding behavior, B8, or the B4-to-B8 inhibitory connection. A conductance-based circuit model indicated that reducing the B4-to-B51 synapse, or increasing B51 excitability, mediated the OC phenotype more effectively than did decreasing B4 excitability. We combined these modifications to examine whether they could act synergistically. Combinations including B51 synergistically enhanced feeding. Taken together, these results suggest modifications of diverse loci work synergistically to mediate OC and that some neurons are well suited to work synergistically with plasticity in other loci.SIGNIFICANCE STATEMENT The ways in which synergism of diverse plasticity loci mediate the change in motor patterns in operant conditioning (OC) are poorly understood. Here, we found that OC was in part mediated by decreasing the intrinsic excitability of a critical neuron of Aplysia feeding behavior, and specifically reducing the strength of one of its inhibitory connections that targets a key decision-making neuron. A conductance-based computational model indicated that the known plasticity loci showed a surprising level of synergism to mediate the behavioral changes associated with OC. These results highlight the importance of understanding the diversity, specificity and synergy among different types of plasticity that encode memory. Also, because OC in Aplysia is mediated by dopamine (DA), the present study provides insights into specific and synergistic mechanisms of DA-mediated reinforcement of behaviors.
Assuntos
Condicionamento Operante/fisiologia , Modelos Neurológicos , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Animais , Aplysia , Simulação por ComputadorRESUMO
The protostome leucokinin (LK) signaling system, including LK peptides and their G protein-coupled receptors, has been characterized in several species. Despite the progress, molecular mechanisms governing LK peptide-receptor interactions remain to be elucidated. Previously, we identified a precursor protein for Aplysia leucokinin-like peptides (ALKs) that contains the greatest number of amidated peptides among LK precursors in all species identified so far. Here, we identified the first ALK receptor from Aplysia, ALKR. We used cell-based IP1 activation assays to demonstrate that two ALK peptides with the most copies, ALK1 and ALK2, activated ALKR with high potencies. Other endogenous ALK-derived peptides bearing the FXXWX-amide motif also activated ALKR to various degrees. Our examination of cross-species activity of ALKs with the Anopheles LK receptor was consistent with a critical role for the FXXWX-amide motif in receptor activity. Furthermore, we showed, through alanine substitution of ALK1, the highly conserved phenylalanine (F), tryptophan (W), and C-terminal amidation were each essential for receptor activation. Finally, we used an artificial intelligence-based protein structure prediction server (Robetta) and Autodock Vina to predict the ligand-bound conformation of ALKR. Our model predicted several interactions (i.e., hydrophobic interactions, hydrogen bonds, and amide-pi stacking) between ALK peptides and ALKR, and several of our substitution and mutagenesis experiments were consistent with the predicted model. In conclusion, our results provide important information defining possible interactions between ALK peptides and their receptors. The workflow utilized here may be useful for studying other ligand-receptor interactions for a neuropeptide signaling system, particularly in protostomes.