RESUMO
Phosphorylation of the MAPK family member extracellular signal-regulated kinase (ERK) is required to induce long-term synaptic plasticity, but little is known about its persistence. We examined ERK activation by three protocols that induce long-term synaptic facilitation (LTF) of the Aplysia sensorimotor synapse - the standard protocol (five 5-min pulses of 5-HT with interstimulus intervals (ISIs) of 20 min), the enhanced protocol (five pulses with irregular ISIs, which induces greater and longer-lasting LTF) and the two-pulse protocol (two pulses with ISI 45 min). Immunofluorescence revealed complex ERK activation. The standard and two-pulse protocols immediately increased active, phosphorylated ERK (pERK), which decayed within 5 h. A second wave of increased pERK was detected 18 h post-treatment for all protocols. This late phase was blocked by inhibitors of protein kinase A, TrkB and TGF-ß. These results suggest that complex interactions among kinase pathways and growth factors contribute to the late increase of pERK. ERK activity returned to basal 24 h after the standard or two-pulse protocols, but remained elevated 24 h for the enhanced protocol. This 24-h elevation was also dependent on PKA and TGF-ß, and partly on TrkB. These results begin to characterize long-lasting ERK activation, plausibly maintained by positive feedback involving growth factors and PKA, that appears essential to maintain LTF and LTM. Because many processes involved in LTF and late LTP are conserved among Aplysia and mammals, these findings highlight the importance of examining the dynamics of kinase cascades involved in vertebrate long-term memory.
RESUMO
Empirical and computational methods were combined to examine whether individual or dual-drug treatments can restore the deficit in long-term synaptic facilitation (LTF) of the Aplysia sensorimotor synapse observed in a cellular model of Coffin-Lowry syndrome (CLS). The model was produced by pharmacological inhibition of p90 ribosomal S6 kinase (RSK) activity. In this model, coapplication of an activator of the mitogen-activated protein kinase (MAPK) isoform ERK and an activator of protein kinase A (PKA) resulted in enhanced phosphorylation of RSK and enhanced LTF to a greater extent than either drug alone and also greater than their additive effects, which is termed synergism. The extent of synergism appeared to depend on another MAPK isoform, p38 MAPK. Inhibition of p38 MAPK facilitated serotonin (5-HT)-induced RSK phosphorylation, indicating that p38 MAPK inhibits activation of RSK. Inhibition of p38 MAPK combined with activation of PKA synergistically activated both ERK and RSK. Our results suggest that cellular models of disorders that affect synaptic plasticity and learning, such as CLS, may constitute a useful strategy to identify candidate drug combinations, and that combining computational models with empirical tests of model predictions can help explain synergism of drug combinations.
Assuntos
Síndrome de Coffin-Lowry , Proteínas Quinases Dependentes de AMP Cíclico , Plasticidade Neuronal , Proteínas Quinases p38 Ativadas por Mitógeno , Humanos , Síndrome de Coffin-Lowry/fisiopatologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Plasticidade Neuronal/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Serotonina/farmacologiaRESUMO
Kinases play critical roles in synaptic and neuronal changes involved in the formation of memory. However, significant gaps exist in the understanding of how interactions among kinase pathways contribute to the mechanistically distinct temporal domains of memory ranging from short-term memory to long-term memory (LTM). Activation of protein kinase A (PKA) and mitogen-activated protein kinase (MAPK)-ribosomal S6 kinase (RSK) pathways are critical for long-term enhancement of neuronal excitability (LTEE) and long-term synaptic facilitation (LTF), essential processes in memory formation. This study provides new insights into how these pathways contribute to the temporal domains of memory, using empirical and computational approaches. Empirical studies of Aplysia sensory neurons identified a positive feedforward loop in which the PKA and ERK pathways converge to regulate RSK, and a negative feedback loop in which p38 MAPK inhibits the activation of ERK and RSK. A computational model incorporated these findings to simulate the dynamics of kinase activity produced by different stimulus protocols and predict the critical roles of kinase interactions in the dynamics of these pathways. These findings may provide insights into the mechanisms underlying aberrant synaptic plasticity observed in genetic disorders such as RASopathies and Coffin-Lowry syndrome.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células Receptoras Sensoriais/citologia , Serotonina/farmacologia , Animais , Aplysia , Células Cultivadas , Pesquisa Empírica , Retroalimentação Fisiológica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Proteínas Quinases S6 Ribossômicas/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismoRESUMO
Multiple kinases converge on the transcription factor cAMP response element-binding protein (CREB) to enhance the expression of proteins essential for long-term synaptic plasticity and memory. The p90 ribosomal S6 kinase (RSK) is one of these kinases, although its role is poorly understood. The present study exploited the technical advantages of the Aplysia sensorimotor culture system to examine the role of RSK in long-term synaptic facilitation (LTF) and long-term enhancement of neuronal excitability (LTEE), two correlates of long-term memory (LTM). Inhibition of RSK expression or RSK activity both significantly reduced CREB1 phosphorylation, LTF, and LTEE, suggesting RSK is required for learning-related synaptic plasticity and enhancement in neuronal excitability. In addition, knock down of RSK by RNAi in Aplysia sensory neurons impairs LTF, suggesting that this may be a useful single-cell system to study aspects of defective synaptic plasticity in Coffin-Lowry Syndrome (CLS), a cognitive disorder that is caused by mutations in rsk2 and associated with deficits in learning and memory. We found that the impairments in LTF and LTEE can be rescued by a computationally designed spaced training protocol, which was previously demonstrated to augment normal LTF and LTM.
Assuntos
Aplysia/fisiologia , Memória de Longo Prazo/fisiologia , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Células Receptoras Sensoriais/citologia , Animais , Aplysia/metabolismo , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Plasticidade Neuronal , Fosforilação , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Células Receptoras Sensoriais/metabolismo , Serotonina/farmacologiaRESUMO
Developing treatment strategies to enhance memory is an important goal of neuroscience research. Activation of multiple biochemical signaling cascades, such as the protein kinase A (PKA) and extracellular signal-regulated kinase (ERK) pathways, is necessary to induce long-term synaptic facilitation (LTF), a correlate of long-term memory (LTM). Previously, a computational model was developed which correctly predicted a novel enhanced training protocol that augmented LTF by searching for the protocol with maximal overlap of PKA and ERK activation. The present study focused on pharmacological approaches to enhance LTF. Combining an ERK activator, NSC, and a PKA activator, rolipram, enhanced LTF to a greater extent than did either drug alone. An even greater increase in LTF occurred when rolipram and NSC were combined with the Enhanced protocol. These results indicate superior memory can be achieved by enhanced protocols that take advantage of the structure and dynamics of the biochemical cascades underlying memory formation, used in conjunction with combinatorial pharmacology.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Potenciação de Longa Duração/fisiologia , Células Receptoras Sensoriais/metabolismo , Animais , Aplysia , Proteína de Ligação a CREB/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Ativadores de Enzimas/farmacologia , Gânglios dos Invertebrados/citologia , Potenciação de Longa Duração/efeitos dos fármacos , Microscopia Confocal , Inibidores da Fosfodiesterase 4/farmacologia , Fosforilação/efeitos dos fármacos , Rolipram/farmacologia , Células Receptoras Sensoriais/efeitos dos fármacos , Serotonina/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Memory impairment is often associated with disrupted regulation of gene induction. For example, deficits in cAMP response element-binding protein (CREB) binding protein (CBP; an essential cofactor for activation of transcription by CREB) impair long-term synaptic plasticity and memory. Previously, we showed that small interfering RNA (siRNA)-induced knockdown of CBP in individual sensory neurons significantly reduced levels of CBP and impaired 5-HT-induced long-term facilitation (LTF) in sensorimotor cocultures from Aplysia. Moreover, computational simulations of the biochemical cascades underlying LTF successfully predicted training protocols that restored LTF following CBP knockdown. We examined whether simulations could also predict a training protocol that restores LTF impaired by siRNA-induced knockdown of the transcription factor CREB1. Simulations based on a previously described model predicted rescue protocols that were specific to CREB1 knockdown. Empirical studies demonstrated that one of these rescue protocols partially restored impaired LTF. In addition, the effectiveness of the rescue protocol was enhanced by pretreatment with rolipram, a selective cAMP phosphodiesterase inhibitor. These results provide further evidence that computational methods can help rescue disruptions in signaling cascades underlying memory formation. Moreover, the study demonstrates that the effectiveness of computationally designed training protocols can be enhanced with complementary pharmacological approaches.
Assuntos
Proteína de Ligação a CREB/metabolismo , Potenciação de Longa Duração/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Sinapses/efeitos dos fármacos , Animais , Aplysia/citologia , Biofísica , Proteína de Ligação a CREB/antagonistas & inibidores , Técnicas de Cocultura , Simulação por Computador , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciação de Longa Duração/fisiologia , Modelos Neurológicos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/fisiologia , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/fisiologia , Serotonina/farmacologia , Sinapses/fisiologia , Fatores de TempoRESUMO
At the University of Texas Houston Medical School, a rotational dissection system was introduced to improve coordination between the Gross Anatomy and the Introduction to Clinical Medicine (ICM) courses. Six students were assigned to each cadaver and divided into two teams. For each laboratory, one team was assigned to dissect and the other to attend ICM or study independently. For the next laboratory, the assignments were reversed. At the start of the session, the team that had dissected previously spent 30 minutes teaching the other team. In 2012, the students were given three traditional practical examinations with 50 questions drawn equally from each laboratory. Students also completed three mid-course evaluations. There were no significant differences in overall performance between the two teams. Nevertheless, we wanted to determine how well individual students identified structures they had dissected compared with those they had not. For dissected structures, the mean percent correct was 80.0 ± 13.0 (mean ± standard deviation), and for undissected structures, it was 78.3 ± 14.1. The difference was small, but statistically significant (P = 0.0007). Although this result validated the concerns expressed by some students, it did not appear that a change in the system was justified. Students were generally enthusiastic about the opportunity to learn clinical skills in the first semester of medical school, and 91-96% of the students agreed that learning anatomy at the same time helped them understand the physical examination exercises in ICM.
Assuntos
Anatomia/educação , Dissecação/educação , Educação de Graduação em Medicina/métodos , Ensino/métodos , Cadáver , Compreensão , Currículo , Avaliação Educacional , Escolaridade , Feminino , Humanos , Aprendizagem , Masculino , Avaliação de Programas e Projetos de Saúde , Faculdades de Medicina , Estudantes de Medicina , Inquéritos e Questionários , Texas , Adulto JovemRESUMO
Doxorubicin (DOX) is an anthracycline used widely for cancer chemotherapy. Its primary mode of action appears to be topoisomerase II inhibition, DNA cleavage, and free radical generation. However, in non-neuronal cells, DOX also inhibits the expression of dual-specificity phosphatases (also referred to as MAPK phosphatases) and thereby inhibits the dephosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAPK), two MAPK isoforms important for long-term memory (LTM) formation. Activation of these kinases by DOX in neurons, if present, could have secondary effects on cognitive functions, such as learning and memory. The present study used cultures of rat cortical neurons and sensory neurons (SNs) of Aplysia to examine the effects of DOX on levels of phosphorylated ERK (pERK) and phosphorylated p38 (p-p38) MAPK. In addition, Aplysia neurons were used to examine the effects of DOX on long-term enhanced excitability, long-term synaptic facilitation (LTF), and long-term synaptic depression (LTD). DOX treatment led to elevated levels of pERK and p-p38 MAPK in SNs and cortical neurons. In addition, it increased phosphorylation of the downstream transcriptional repressor cAMP response element-binding protein 2 in SNs. DOX treatment blocked serotonin-induced LTF and enhanced LTD induced by the neuropeptide Phe-Met-Arg-Phe-NH2. The block of LTF appeared to be attributable to overriding inhibitory effects of p-p38 MAPK, because LTF was rescued in the presence of an inhibitor (SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole]) of p38 MAPK. These results suggest that acute application of DOX might impair the formation of LTM via the p38 MAPK pathway.
Assuntos
Doxorrubicina/farmacologia , Potenciação de Longa Duração/efeitos dos fármacos , Serotonina/farmacologia , Sinapses/efeitos dos fármacos , Inibidores da Topoisomerase II/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Aplysia , Células Cultivadas , Córtex Cerebral/citologia , Técnicas de Cocultura , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Depressão Sináptica de Longo Prazo , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/fisiologia , Fosforilação/efeitos dos fármacos , Ratos , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/fisiologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Mutations in the gene encoding CREB-binding protein (CBP) cause deficits in long-term plasticity, learning, and memory. Here, long-term synaptic facilitation (LTF) at Aplysia sensorimotor synapses in cell culture was used as a model system to investigate methods for overcoming deficits in LTF produced by a CBP knockdown. Injecting CBP-siRNA into individual sensory neurons reduced CBP levels and impaired LTF produced by a standard protocol of five 5-min pulses of serotonin (5-HT) delivered at 20 min interstimulus intervals. A computational model, which simulated molecular processes underlying LTF induction, predicted a rescue protocol of five pulses of 5-HT at non-uniform interstimulus intervals that overcame the consequences of reduced CBP and restored LTF. These results suggest that complementary empirical and computational studies can identify methods for ameliorating impairments of learning attributable to molecular lesions.
Assuntos
Proteína de Ligação a CREB/metabolismo , Simulação por Computador , Modelos Neurológicos , Plasticidade Neuronal/fisiologia , Sinapses/fisiologia , Análise de Variância , Animais , Aplysia , Biofísica , Proteína de Ligação a CREB/genética , Células Cultivadas , Técnicas de Cocultura , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Neurônios Motores/citologia , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/genética , Valor Preditivo dos Testes , RNA Interferente Pequeno/farmacologia , Células Receptoras Sensoriais/citologia , Serotonina/farmacologia , Sinapses/efeitos dos fármacosRESUMO
Serotonin (5-HT)-induced long-term facilitation (LTF) of the Aplysia sensorimotor synapse depends on enhanced gene expression and protein synthesis, but identification of the genes whose expression and regulation are necessary for LTF remains incomplete. In this study, we found that one such gene is synapsin, which encodes a synaptic vesicle-associated protein known to regulate short-term synaptic plasticity. Both synapsin mRNA and protein levels were increased by 5-HT. Upregulation of synapsin protein occurred in presynaptic sensory neurons at neurotransmitter release sites. To investigate the molecular mechanisms underlying synapsin regulation, we cloned the promoter region of Aplysia synapsin, and found that the synapsin promoter contained a cAMP response element (CRE), raising the possibility that the transcriptional activator CRE-binding protein 1 (CREB1) mediates 5-HT-induced regulation of synapsin. Indeed, binding of CREB1 to the synapsin promoter was increased following treatment with 5-HT. Furthermore, increased acetylation of histones H3 and H4 and decreased association of histone deacetylase 5 near the CRE site are consistent with transcriptional activation by CREB1. RNA interference (RNAi) targeting synapsin mRNA blocked the 5-HT-induced increase in synapsin protein levels and LTF; in the absence of 5-HT treatment, basal synapsin levels were unaffected. These results indicate that the 5-HT-induced regulation of synapsin levels is necessary for LTF and that this regulation is part of the cascade of synaptic events involved in the consolidation of memory.
Assuntos
Potenciação de Longa Duração/fisiologia , Serotonina/metabolismo , Sinapses/metabolismo , Sinapsinas/metabolismo , Acetilação , Animais , Aplysia/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Histonas/genética , Histonas/metabolismo , Regiões Promotoras Genéticas , Serotonina/genética , Sinapses/genética , Sinapsinas/genéticaRESUMO
Learning and memory are influenced by the temporal pattern of training stimuli. However, the mechanisms that determine the effectiveness of a particular training protocol are not well understood. We tested the hypothesis that the efficacy of a protocol is determined in part by interactions among biochemical cascades that underlie learning and memory. Previous findings suggest that the protein kinase A (PKA) and extracellular signal-regulated kinase (ERK) cascades are necessary to induce long-term synaptic facilitation (LTF) in Aplysia, a neuronal correlate of memory. We developed a computational model of the PKA and ERK cascades and used it to identify a training protocol that maximized PKA and ERK interactions. In vitro studies confirmed that the protocol enhanced LTF. Moreover, the protocol enhanced the levels of phosphorylation of the transcription factor CREB1. Behavioral training confirmed that long-term memory also was enhanced by the protocol. These results illustrate the feasibility of using computational models to design training protocols that improve memory.
Assuntos
Simulação por Computador , Aprendizagem/fisiologia , Modelos Biológicos , Análise de Variância , Animais , Aplysia , Proteína de Ligação a CREB/metabolismo , Células Cultivadas , Técnicas de Cocultura , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Estimulação Elétrica , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Lateralidade Funcional , Gânglios dos Invertebrados/citologia , Humanos , Aprendizagem/efeitos dos fármacos , Potenciação de Longa Duração/fisiologia , Neurônios Motores/fisiologia , Células Receptoras Sensoriais/fisiologia , Serotonina/farmacologia , Transdução de SinaisRESUMO
Accumulating evidence suggests that the transcriptional activator cAMP response element-binding protein 1 (CREB1) is important for serotonin (5-HT)-induced long-term facilitation (LTF) of the sensorimotor synapse in Aplysia. Moreover, creb1 is among the genes activated by CREB1, suggesting a role for this protein beyond the induction phase of LTF. The time course of the requirement for CREB1 synthesis in the consolidation of long-term facilitation was examined using RNA interference techniques in sensorimotor cocultures. Injection of CREB1 small-interfering RNA (siRNA) immediately or 10 h after 5-HT treatment blocked LTF when measured at 24 and 48 h after treatment. In contrast, CREB1 siRNA did not block LTF when injected 16 h after 5-HT treatment. These results demonstrate that creb1 expression must be sustained for a relatively long time to support the consolidation of LTF. In addition, LTF is also accompanied by a long-term increase in the excitability (LTE) of sensory neurons (SNs). Because LTE was observed in the isolated SN after 5-HT treatment, this long-term change was intrinsic to that element of the circuit. LTE was blocked when CREB1 siRNA was injected into isolated SNs immediately after 5-HT treatment. These data suggest that 5-HT-induced CREB1 synthesis is required for consolidation of both LTF and LTE.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Potenciação de Longa Duração/fisiologia , Células Receptoras Sensoriais/fisiologia , Sinapses/fisiologia , Animais , Aplysia , Células Cultivadas , Técnicas de Cocultura , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Eletrofisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , RNA Interferente PequenoRESUMO
Long-term memory and plasticity, including long-term synaptic facilitation (LTF) of the Aplysia sensorimotor synapse, depend on the activation of transcription factors that regulate genes necessary for synaptic plasticity. In the present study we found that treatment with 5-HT and behavioral training produce biphasic changes in the expression of CREB2, a transcriptional repressor. An immediate increase in CREB2 protein was followed by a subsequent decrease. The effects of these treatments persist for at least 24 h and are observed in isolated sensory neurons. This study suggests that the dynamics of CREB2 expression could contribute to the consolidation of memory.
Assuntos
Condicionamento Clássico/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Proteínas Repressoras/metabolismo , Serotonina/farmacologia , Animais , Anticorpos/farmacologia , Aplysia , Comportamento Animal , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Estimulação Elétrica/métodos , Lateralidade Funcional , Gânglios dos Invertebrados/citologia , Regulação da Expressão Gênica/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/imunologia , Dinâmica não Linear , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Fatores de TempoRESUMO
Synapsin is a synaptic vesicle-associated protein implicated in the regulation of vesicle trafficking and transmitter release, but its role in heterosynaptic plasticity remains elusive. Moreover, contradictory results have obscured the contribution of synapsin to homosynaptic plasticity. We previously reported that the neuromodulator serotonin (5-HT) led to the phosphorylation and redistribution of Aplysia synapsin, suggesting that synapsin may be a good candidate for the regulation of vesicle mobilization underlying the short-term synaptic plasticity induced by 5-HT. This study examined the role of synapsin in homosynaptic and heterosynaptic plasticity. Overexpression of synapsin reduced basal transmission and enhanced homosynaptic depression. Although synapsin did not affect spontaneous recovery from depression, it potentiated 5-HT-induced dedepression. Computational analysis showed that the effects of synapsin on plasticity could be adequately simulated by altering the rate of Ca(2+)-dependent vesicle mobilization, supporting the involvement of synapsin not only in homosynaptic but also in heterosynaptic forms of plasticity by regulating vesicle mobilization.
Assuntos
Aplysia/fisiologia , Serotonina/farmacologia , Sinapses/efeitos dos fármacos , Sinapses/fisiologia , Sinapsinas/fisiologia , Animais , Técnicas de Cocultura , Simulação por Computador , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Eletrofisiologia , Imunofluorescência , Corantes Fluorescentes , Neurônios Motores/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Neurônios Aferentes/efeitos dos fármacos , Técnicas de Patch-Clamp , Plasmídeos/genética , Compostos de Piridínio , Compostos de Amônio Quaternário , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sinapsinas/genética , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/ultraestruturaRESUMO
Repetitive, unilateral stimulation of Aplysia induces long-term sensitization (LTS) of ipsilaterally elicited siphon-withdrawal responses. Whereas some morphological effects of training appear only on ipsilateral sensory neurons, others appear bilaterally. We tested the possibility that contralateral morphological modifications may have functional significance. Therefore, we examined whether LTS training primes subsequent sensitization. Twenty-four hours after LTS training the effects of brief shock treatment (BST) were examined. BST failed to sensitize animals that had previously received either 4-d control treatment or 4-d ipsilateral LTS training. In contrast, BST did sensitize animals that had previously received 4-d contralateral LTS training, suggesting the presence of a latent trace that primes the animal for further learning.
Assuntos
Aplysia/fisiologia , Aprendizagem/fisiologia , Memória/fisiologia , Neurônios Aferentes/fisiologia , Animais , Lateralidade Funcional , Fatores de TempoRESUMO
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.
Assuntos
Potenciais de Ação/fisiologia , Aplysia/citologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Neurônios Aferentes/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/efeitos da radiação , Animais , Butadienos/farmacologia , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Imunofluorescência/métodos , Neurônios Aferentes/fisiologia , Nitrilas/farmacologia , Fatores de Tempo , Fator de Crescimento Transformador beta1RESUMO
Neurite outgrowth is a process commonly thought to contribute to long-term learning by formation of new synaptic contacts. The behavioral effects of long-term sensitization training in Aplysia were restricted to the trained side of the animal as were changes in strength of the sensorimotor synapse. In contrast, training produced varicosity formation on both sides of the animal. Appositions with follower neurons, however, were enhanced only on the trained side. The dissociation of structural and functional correlates suggests that key regulatory steps are downstream from outgrowth, possibly in the targeting of new processes and activation of new synapses.
Assuntos
Aplysia/anatomia & histologia , Aplysia/fisiologia , Memória/fisiologia , Fenômenos Fisiológicos do Sistema Nervoso , Sistema Nervoso/crescimento & desenvolvimento , Neurônios/fisiologia , Animais , Condicionamento Clássico/fisiologia , Gânglios dos Invertebrados/citologia , Gânglios dos Invertebrados/fisiologia , Microscopia Confocal , Sistema Nervoso/citologia , Neuritos/fisiologia , Plasticidade Neuronal , Neurônios/ultraestrutura , Sinapses/fisiologiaRESUMO
Present models of long-term sensitization in Aplysia californica indicate that the enhanced behavioral response is due, at least in part, to outgrowth of sensory neurons mediating defensive withdrawal reflexes. Presumably, this outgrowth strengthens pre-existing connections by formation of new synapses with follower neurons. However, the relationship between the number of sensorimotor contacts and the physiological strength of the connection has never been examined in intact ganglia. As a first step in addressing this issue, we used confocal microscopy to examine sites of contact between sensory and motor neurons in naive animals. Our results revealed relatively few contacts between physiologically connected cells. In addition, the number of contact sites was proportional to the amplitude of the EPSP elicited in the follower motor neuron by direct stimulation of the sensory neuron. This is the first time such a correlation has been observed in the central nervous system. Serotonin is the neurotransmitter most closely examined for its role in modulating synaptic strength at the sensorimotor synapse. However, the structural relationship of serotonergic processes and sensorimotor synapses has never been examined. Surprisingly, serotonergic processes usually made contact with sensory and motor neurons at sites located relatively distant from the sensorimotor synapse. This result implies that heterosynaptic regulation is due to nondirected release of serotonin into the neuropil.
Assuntos
Gânglios dos Invertebrados/citologia , Neurônios Motores , Vias Neurais/anatomia & histologia , Neurônios Aferentes , Serotonina , Sinapses , Animais , Aplysia , Potenciais Pós-Sinápticos Excitadores , Gânglios dos Invertebrados/anatomia & histologia , Gânglios dos Invertebrados/fisiologia , Imuno-Histoquímica , Microscopia ConfocalRESUMO
Withdrawal reflexes of Aplysia are mediated in part by a monosynaptic circuit of sensory (SN) and motor (MN) neurons. A brief high-frequency burst of spikes in the SN produces excitatory postsynaptic potentials (EPSPs) that rapidly decrease in amplitude during the burst of activity. It is generally believed that this and other (i.e., low-frequency) forms of homosynaptic depression are entirely caused by presynaptic mechanisms (e.g., depletion of releasable transmitter). The present study examines the contribution that desensitization of postsynaptic glutamate receptors makes to homosynaptic depression. Bath application of cyclothiazide, an agent that reduces desensitization of non-NMDA glutamate receptors, reduced high-, but not low-frequency synaptic depression. Thus, a postsynaptic mechanism, desensitization of glutamate receptors, can also contribute to homosynaptic depression of sensorimotor synapses.
Assuntos
Depressão Sináptica de Longo Prazo , Neurônios Motores/fisiologia , Neurônios Aferentes/fisiologia , Receptores de AMPA/fisiologia , Reflexo/fisiologia , Animais , Aplysia , Benzotiadiazinas/farmacologia , Regulação para Baixo , Estimulação Elétrica , Eletrofisiologia , Potenciais Pós-Sinápticos Excitadores , Terminações Pré-Sinápticas , Receptores de Glutamato/fisiologia , Transmissão SinápticaRESUMO
Only a small fraction of neurotransmitter-containing synaptic vesicles (SVs), the readily releasable pool, is available for fast Ca(2+)-induced release at any synapse. Most SVs are sequestered at sites away from the plasma membrane and cannot be exocytosed directly. Recruitment of SVs to the releasable pool is thought to be an important component of short-term synaptic facilitation by serotonin (5-HT) at Aplysia sensorimotor synapses. Synapsins are associated with SVs and hypothesized to play a central role in the regulation of SV mobilization in nerve terminals. Aplysia synapsin was cloned to examine its role in synaptic plasticity at the well characterized sensorimotor neuron synapse of this animal. Acute 5-HT treatment of ganglia induced synapsin phosphorylation. Immunohistochemical analyses of cultured Aplysia neurons revealed that synapsin is distributed in distinct puncta in the neurites. These puncta are rapidly dispersed after treatment of the neurons with 5-HT. The dispersion of synapsin puncta by 5-HT was fully reversible after washout of the modulator. Both 5-HT-induced phosphorylation and dispersion of synapsin were mediated, at least in part, by cAMP-dependent protein kinase and mitogen-activated protein kinase. These experiments indicate that synapsin and its regulation by 5-HT may play an important role in the modulation of SV trafficking in short-term synaptic plasticity.