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An in vitro model of delay eyeblink classical conditioning was developed to investigate synaptic plasticity mechanisms underlying acquisition of associative learning. This was achieved by replacing real stimuli, such as an airpuff and tone, with patterned stimulation of the cranial nerves using an isolated brainstem preparation from turtle. Here, our primary findings regarding cellular and molecular mechanisms for learning acquisition using this unique approach are reviewed. The neural correlate of the in vitro eyeblink response is a replica of the actual behavior, and features of conditioned responses (CRs) resemble those observed in behavioral studies. Importantly, it was shown that acquisition of CRs did not require the intact cerebellum, but the appropriate timing did. Studies of synaptic mechanisms indicate that conditioning involves two stages of AMPA receptor (AMPAR) trafficking. Initially, GluA1-containing AMPARs are targeted to synapses followed later by replacement by GluA4 subunits that support CR expression. This two-stage process is regulated by specific signal transduction cascades involving PKA and PKC and is guided by distinct protein chaperones. The expression of the brain-derived neurotrophic factor (BDNF) protein is central to AMPAR trafficking and conditioning. BDNF gene expression is regulated by coordinated epigenetic mechanisms involving DNA methylation/demethylation and chromatin modifications that control access of promoters to transcription factors. Finally, a hypothesis is proposed that learning genes like BDNF are poised by dual chromatin features that allow rapid activation or repression in response to environmental stimuli. These in vitro studies have advanced our understanding of the cellular and molecular mechanisms that underlie associative learning.
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Condicionamiento Clásico , Receptores AMPA , Receptores AMPA/genética , Factor Neurotrófico Derivado del Encéfalo , Regulación de la Expresión Génica , CromatinaRESUMEN
Social decision-making is critically influenced by neurocircuitries that regulate stress responsiveness. Adaptive choices, therefore, are altered by stress-related neuromodulatory peptide systems, such as corticotropin releasing factor (CRF). Experimental designs that take advantage of ecologically salient fear-inducing stimuli allow for revelation of neural mechanisms that regulate the balance between pro- and anti-stress responsiveness. To accomplish this, we developed a social stress and conditioning protocol, the Stress Alternatives Model (SAM), that utilizes a simple dichotomous choice, and produces distinctive behavioral phenotypes (Escape or Stay). The experiments involve repeated social aggression, a potent unconditioned stimulus (US), from a novel larger conspecific (a 3X larger Rainbow trout). Prior to the social interaction, the smaller test fish is presented with an auditory conditioning stimulus (water off = CS). During the social aggression, an escape route is available, but is only large enough for the smaller test animal. Surprisingly, although the new aggressor provides vigorous attacks each day, only 50% of the test fish choose Escape. Stay fish, treated with the CRF1 antagonist antalarmin, a potent anxiolytic drug, on day 4, promotes Escape behavior for the last 4 days of the SAM protocol. The results suggest that the decision to Escape, required a reduction in stress reactivity. The Stay fish that chose Escape following anxiolytic treatment, learned how to use the escape route prior to stress reduction, as the Escape latency in these fish was significantly faster than first time escapers. In Escape fish, the use of the escape route is learned over several days, reducing the Escape latency over time in the SAM. Fear conditioning (water off + aggression) resulted in elevated hippocampal (DL) Bdnf mRNA levels, with coincident reduction in the AMPA receptor subunit Glua1 expression, a result that is reversed following a one-time treatment (during SAM aggression on day 4) with the anxiolytic CRF1 receptor antagonist antalarmin.
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Ansiolíticos , Animales , Ansiolíticos/farmacología , Hormona Liberadora de Corticotropina/metabolismo , Aprendizaje , Miedo/fisiología , Receptores de Hormona Liberadora de Corticotropina , Expresión GénicaRESUMEN
One of the longstanding goals of the field of neuroscience is to understand the neural control of behavior in both invertebrate and vertebrate species. A series of early discoveries showed that certain motor patterns like locomotion could be generated by neuronal circuits without sensory feedback or descending control systems. These were called fictitious, or "fictive," motor programs because they could be expressed by neurons in the absence of movement. This finding led investigators to isolate central nervous system tissue and maintain it in a dish in vitro to better study mechanisms of motor pattern generation. A period of rapid development of in vitro preparations from invertebrate species that could generate fictive motor programs from the activity of central pattern-generating circuits (CPGs) emerged that was gradually followed by the introduction of such preparations from vertebrates. Here, I will review some of the notable in vitro preparations from both mammalian and nonmammalian vertebrate species developed to study the neural circuits underlying a variety of complex behaviors. This approach has been instrumental in delineating not only the cellular substrates underlying locomotion, respiration, scratching, and other behaviors, but also mechanisms underlying the modifiability of motor pathways through synaptic plasticity. In vitro preparations have had a significant impact on the field of motor systems neuroscience and the expansion of our understanding of how nervous systems control behavior. The field is ready for further advancement of this approach to explore neural substrates for variations in behavior generated by social and seasonal context, and the environment.
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Locomoción , Neuronas Motoras , Animales , Sistema Nervioso Central , Mamíferos , Neuronas Motoras/fisiología , Plasticidad Neuronal , VertebradosRESUMEN
Research demonstrates that the neural mechanisms underlying synaptic plasticity and learning and memory involve mobilization of AMPA-type neurotransmitter receptors at glutamatergic synaptic contacts, and that these mechanisms are targeted during neurodegenerative disease. Strengthening neural transmission occurs with insertion of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) into synapses while weakening results from receptor withdrawal. A key player in the trafficking of AMPARs during plasticity and learning is the brain-derived neurotrophic factor (BDNF) signaling system. BDNF is a neurotrophic factor that supports neuronal growth and is required for learning and memory. Significantly, a primary feature of many neurodegenerative diseases is a reduction in BDNF protein as well as disrupted neuronal surface expression of synaptic AMPARs. The resulting weakening of synaptic contacts leads to synapse loss and neuronal degeneration that underlies the cognitive impairment and dementia observed in patients with progressive neurodegenerative disease such as Alzheimer's. In the face of these data, one therapeutic approach is to increase BDNF bioavailability in brain. While this has been met with significant challenges, the results of the research have been promising. In spite of this, there are currently no clinical trials to test many of these findings on patients. Here, research showing that BDNF drives AMPARs to synapses, AMPAR trafficking is essential for synaptic plasticity and learning, and that neurodegenerative disease results in a significant decline in BDNF will be reviewed. The aim is to draw attention to the need for increasing patient-directed clinical studies to test the possible benefits of increasing levels of neurotrophins, specifically BDNF, to treat brain disorders. Much is known about the cellular mechanisms that underlie learning and memory in brain. It can be concluded that signaling by neurotrophins like BDNF and AMPA-type glutamate receptor synaptic trafficking are fundamental to these processes. Data from animal models and patients reveal that these mechanisms are adversely targeted during neurodegenerative disease and results in memory loss and cognitive decline. A brief summary of our understanding of these mechanisms indicates that it is time to apply this knowledge base directly to development of therapeutic treatments that enhance neurotrophins for brain disorders in patient populations.
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Encefalopatías , Enfermedades Neurodegenerativas , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Proteínas Portadoras , Humanos , Plasticidad Neuronal/fisiología , Receptores AMPA/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol PropiónicoRESUMEN
We previously argued that the neuroscience community has a role in environmental conservation because protection of biodiversity and the specialized behavioral adaptions of animals is essential to understanding brain structure and function. Preserving biodiversity and the natural world is also linked to human mental health and broadens our insight on the origins of psychiatric disorders like stress, anxiety, and depression. The study of neuroscience has become a global scientific pursuit that involves thousands of researchers and has an economic impact in the billions of dollars. As a group of biomedical research scientists, neuroscientists have the knowledge base and public credibility to convincingly promote sustainable environmental actions and policies. Here, we outline several key areas in which we as a neuroscience academic community can participate to preserve a rich global biodiversity and confront the environmental crises that lie before us.
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Biodiversidad , Conservación de los Recursos Naturales , Animales , Humanos , PolíticasRESUMEN
Alternative splicing of genes in the central nervous system is ubiquitous and utilizes many different mechanisms. Splicing generates unique transcript or protein isoforms of the primary gene that result in shortened, lengthened, or reorganized products that may have distinct functions from the parent gene. Learning and memory genes respond selectively to a variety of environmental stimuli and have evolved a number of complex mechanisms for transcriptional regulation to act rapidly and flexibly to environmental demands. Their patterns of expression, however, are incompletely understood. Many activity-inducible genes generate transcripts by alternative splicing that have an unknown physiological or behavioral function. One such gene codes for the protein brain-derived neurotrophic factor (BDNF). BDNF is a neurotrophin whose expression is essential for cellular growth, synaptogenesis, and synaptic plasticity. It is an important model gene because of its complex structure and the variety of transcriptional mechanisms it displays for expression in response to external stimuli. Some of these are unexpected, or non-canonical, transcriptional control mechanisms that require further exploration in an activity-dependent context. In this review, a comparative genomics approach is taken to highlight the different forms of BDNF gene transcription including potential autoregulatory mechanisms. Modes of BDNF control have general implications for understanding the origins of several neurological disorders that are associated with reduced BDNF function.
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Factor Neurotrófico Derivado del Encéfalo/genética , Regulación de la Expresión Génica , Genómica , Enfermedades del Sistema Nervioso/genética , Empalme Alternativo/genética , Secuencia de Aminoácidos , Animales , Factor Neurotrófico Derivado del Encéfalo/química , Humanos , Especificidad de la EspecieRESUMEN
The vertebrate brain-derived neurotrophic factor (BDNF) gene produces a number of alternatively spliced transcripts only some of which generate the BDNF protein required for synaptic plasticity and learning. Many of the transcripts are uncharacterized and are of unknown biological significance. Previously, we described alternative splicing within the protein-coding sequence of the BDNF gene in the pond turtle (tBDNF) that generates a functionally distinct truncated protein isoform (trcBDNF) that is regulated during a neural correlate of eyeblink classical conditioning in ex vivo brainstem preparations. We hypothesized that trcBDNF has a dominant negative function because of its anticorrelated expression pattern compared to its full-length BDNF counterpart. The data presented here suggests that trcBDNF functions as a transcriptional repressor of a conditioning-inducible downstream tBDNF promoter that controls expression of full-length BDNF required for learning. First, expression of full-length transcripts is negatively correlated with trcBDNF; transcripts are inhibited when endogenous trcBDNF is ectopically induced and expressed when trcBDNF is inhibited. Second, ChIP-qPCR assays of a recombinant trcBDNF protein, RtrcBDNF, show strong binding to the downstream tBDNF exon III promoter that corresponds with inhibition of conditioning. Third, deletions of the C-terminus of RtrcBDNF result in inhibition of promoter binding and conditioning acquisition when a tropomyosin receptor kinase B (TrkB) binding site is accounted for. Finally, microinjection of RtrcBDNF directly into brainstem preparations inhibits conditioning. These data reveal a new mechanism of activity-dependent BDNF transcriptional regulation and suggest that BDNF is an autoregulatory gene. How generalizable this mechanism is across plasticity genes remains to be elucidated.
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Empalme Alternativo , Factor Neurotrófico Derivado del Encéfalo/genética , Condicionamiento Clásico , Tortugas/metabolismo , Animales , Tronco Encefálico/metabolismo , Tronco Encefálico/fisiología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Tortugas/genética , Tortugas/fisiologíaRESUMEN
Plasticity and learning genes require regulatory mechanisms that have the flexibility to respond to a variety of sensory stimuli to generate adaptive behavioral responses. The immediate early gene (IEG) activity-regulated cytoskeleton-associated protein (ARC) is rapidly induced not only by neuronal stimulation but also during a variety of learning tasks. How ARC is regulated in response to complex stimuli during associative learning remains to be fully detailed. Here, we characterized the structure of the ARC gene in the pond turtle and mechanisms of its transcriptional activation during a neural correlate of eyeblink classical conditioning. The tARC gene is regulated in part by the presence of paused polymerase (RNAPII) that is poised at the promoter for rapid gene induction. Conditioning induces permissive chromatin modifications in the tARC promoter that allows binding by the transcription factor cAMP response element-binding protein (CREB) within 5 min of training. During learning acquisition, the pausing factor negative elongation factor (NELF) dissociates from the promoter thereby releasing RNAPII for active transcription. Data additionally suggest that the DNA insulator protein CCCTC-binding factor (CTCF) is required for transcription by mediating a learning-induced interaction of the ARC promoter with an enhancer element. Our study suggests that the learning-inducible IEG tARC utilizes both paused RNAPII and rapid chromatin modifications that allow for dynamic gene responsiveness required when an organism is presented with a variety of environmental stimuli.
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Condicionamiento Clásico/fisiología , Proteínas del Citoesqueleto/genética , Genes Inmediatos-Precoces , Proteínas Inmediatas-Precoces/genética , Aprendizaje/fisiología , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Puente/fisiología , Transcripción Genética , Animales , Sitios de Unión , Parpadeo/fisiología , Factor de Unión a CCCTC/metabolismo , Ensamble y Desensamble de Cromatina , Nervio Coclear/fisiología , AMP Cíclico/fisiología , Proteínas del Citoesqueleto/biosíntesis , Estimulación Eléctrica , Femenino , Regulación de la Expresión Génica , Proteínas Inmediatas-Precoces/biosíntesis , Técnicas In Vitro , Masculino , Proteínas del Tejido Nervioso/biosíntesis , Regiones Promotoras Genéticas/genética , ARN Polimerasa II/metabolismo , Proteínas Recombinantes/metabolismo , Sistemas de Mensajero Secundario , Factores de Transcripción/metabolismo , Nervio Trigémino/fisiología , Tortugas/genética , Tortugas/metabolismoRESUMEN
Use of hypothermia as a means of anesthesia for amphibians and reptiles is prohibited by agencies that establish veterinary guidelines. This has recently been called into question by members of the scientific community based on reviews of published literature. Using pond turtles (Trachemys scripta elegans), hypothermia as a method for anesthesia to precede euthanasia by decapitation was assessed. Turtles were subjected to hypothermia using a cooling followed by freezing protocol. Body temperature measurements ranged between -1 and -2°C while core body temperature was -1°C. Ice crystal formation was never observed. A protective reflex to noxious stimuli, the eyeblink response, was recorded from in vitro brainstem preparations subjected to cold. At 5-6°C, reflex responses were suppressed, demonstrating minimal synaptic transmission in brain circuits above temperatures used for hypothermia induction. These and previous data indicate that a re-evaluation of the use of hypothermia as an anesthetic in amphibians and reptiles is warranted.
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Anestesia/veterinaria , Hipotermia Inducida/veterinaria , Tortugas/fisiología , Anestesia/métodos , Bienestar del Animal , Animales , Frío , Eutanasia Animal , Femenino , MasculinoRESUMEN
MECP2 mutations underlying Rett syndrome cause widespread misregulation of gene expression. Functions for MeCP2 other than transcriptional are not well understood. In an ex vivo brain preparation from the pond turtle Trachemys scripta elegans, an intraexonic splicing event in the brain-derived neurotrophic factor (BDNF) gene generates a truncated mRNA transcript in naïve brain that is suppressed upon classical conditioning. MeCP2 and its partners, splicing factor Y-box binding protein 1 (YB-1) and methylcytosine dioxygenase 1 (Tet1), bind to BDNF chromatin in naïve but dissociate during conditioning; the dissociation correlating with decreased DNA methylation. Surprisingly, conditioning results in new occupancy of BDNF chromatin by DNA insulator protein CCCTC-binding factor (CTCF), which is associated with suppression of splicing in conditioning. Knockdown of MeCP2 shows it is instrumental for splicing and inhibits Tet1 and CTCF binding thereby negatively impacting DNA methylation and conditioning-dependent splicing regulation. Thus, mutations in MECP2 can have secondary effects on DNA methylation and alternative splicing.
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Empalme Alternativo , Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Aprendizaje , Proteína 2 de Unión a Metil-CpG/metabolismo , Oxigenasas de Función Mixta/metabolismo , Tortugas/fisiología , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Cromatina/metabolismo , Condicionamiento Clásico , ADN/metabolismo , Desmetilación , Unión Proteica , Proteína 1 de Unión a la Caja Y/metabolismoRESUMEN
Learning genes in mature neurons are uniquely suited to respond rapidly to specific environmental stimuli. Expression of individual learning genes, therefore, requires regulatory mechanisms that have the flexibility to respond with transcriptional activation or repression to select appropriate physiological and behavioral responses. Among the mechanisms that equip genes to respond adaptively are bivalent domains. These are specific histone modifications localized to gene promoters that are characteristic of both gene activation and repression, and have been studied primarily for developmental genes in embryonic stem cells. In this review, studies of the epigenetic regulation of learning genes in neurons, particularly the brain-derived neurotrophic factor gene (BDNF), by methylation/demethylation and chromatin modifications in the context of learning and memory will be highlighted. Because of the unique function of learning genes in the mature brain, it is proposed that bivalent domains are a characteristic feature of the chromatin landscape surrounding their promoters. This allows them to be "poised" for rapid response to activate or repress gene expression depending on environmental stimuli.
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AMPA receptor (AMPAR) trafficking has emerged as a fundamental concept for understanding mechanisms of learning and memory as well as many neurological disorders. Classical conditioning is a simple and highly conserved form of associative learning. Our studies use an ex vivo brainstem preparation in which to study cellular mechanisms underlying learning during a neural correlate of eyeblink conditioning. Two stages of AMPAR synaptic delivery underlie conditioning utilizing sequential trafficking of GluA1-containing AMPARs early in conditioning followed by replacement with GluA4 subunits later. Subunit-selective trafficking of AMPARs is poorly understood. Here, we focused on identification of auxiliary chaperone proteins that traffic AMPARs. The results show that auxiliary proteins TARPγ8 and GSG1L are colocalized with AMPARs on abducens motor neurons that generate the conditioning. Significantly, TARPγ8 was observed to chaperone GluA1-containing AMPARs during synaptic delivery early in conditioning while GSG1L chaperones GluA4 subunits later in conditioning. Interestingly, TARPγ8 remains at the membrane surface as GluA1 subunits are withdrawn and associates with GluA4 when they are delivered to synapses. These data indicate that GluA1- and GluA4-containing AMPARs are selectively chaperoned by TARPγ8 and GSG1L, respectively. Therefore, sequential subunit-selective trafficking of AMPARs during conditioning is achieved through the timing of their interactions with specific auxiliary proteins.
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Condicionamiento Clásico/fisiología , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Receptores AMPA/metabolismo , Sinapsis/metabolismo , Tortugas/fisiología , Nervio Abducens/citología , Nervio Abducens/fisiología , Animales , Parpadeo , Tronco Encefálico/metabolismo , Membrana Celular/metabolismo , Femenino , Masculino , Neuronas Motoras/metabolismo , Subunidades de Proteína/metabolismo , Transporte de ProteínasRESUMEN
Current trends in neuroscience research have moved toward a reliance on rodent animal models to study most aspects of brain function. Such laboratory-reared animals are highly inbred, have been disengaged from their natural environments for generations and appear to be of limited predictive value for successful clinical outcomes. In this Perspective article, we argue that research on a rich diversity of animal model systems is fundamental to new discoveries in evolutionarily conserved core physiological and molecular mechanisms that are the foundation of human brain function. Analysis of neural circuits across phyla will reveal general computational solutions that form the basis for adaptive behavioral responses. Further, we stress that development of ethoexperimental approaches to improve our understanding of behavioral nuance will help to realign our research strategies with therapeutic goals and improve the translational validity of specific animal models. Finally, we suggest that neuroscience has a role in environmental conservation of habitat and fauna that will preserve and protect the ecological settings that drive species-specific behavioral adaptations. A rich biodiversity will enhance our understanding of human brain function and lead in unpredicted directions for development of therapeutic treatments for neurological disorders.
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MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression posttranscriptionally by interfering with translation of their target mRNAs. Typically, miRNAs bind to the 3' UTRs of mRNAs to induce repression or degradation. Neurotrophins are growth factors in brain required for neuronal survival, synapse formation, and plasticity mechanisms. Neurotrophins are not only regulated by miRNAs, but they in turn regulate miRNA expression. Accumulating data indicate there is a regulatory negative feedback loop between one ubiquitous neurotrophin, brain-derived neurotrophic factor (BDNF), and miRNAs. That is, while BDNF treatment stimulates neuronal miRNA expression, miRNAs generally function to inhibit expression of BDNF. This negative feedback loop is maintained in a state of equilibrium in normal cells. However, in Alzheimer's Disease (AD), a progressive neurodegenerative disorder resulting in memory loss and eventually dementia that is characterized by reduced levels of BDNF in brain, the balance between BDNF and miRNA is shifted toward inhibitory control by miRNAs. Here, we will briefly review the evidence for a positive action of BDNF on miRNA expression and a negative action of miRNAs on BDNF. We propose that the reduction in BDNF that occurs in the AD brain is the result of two independent mechanisms: 1) a failure in the proteolytic conversion of BDNF precursor protein to its functional mature form, and 2) inhibition of BDNF gene expression by miRNAs. The role of miRNAs in BDNF regulation should be considered when developing BDNF-based therapeutic treatments for AD.
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Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Encéfalo/metabolismo , Regulación de la Expresión Génica , MicroARNs/genética , Transducción de Señal , Animales , Factor Neurotrófico Derivado del Encéfalo/farmacología , Regulación hacia Abajo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interferencia de ARN , Transducción de Señal/efectos de los fármacosRESUMEN
Brain-derived neurotrophic factor (BDNF) gene expression critically controls learning and its aberrant regulation is implicated in Alzheimer's disease and a host of neurodevelopmental disorders. The BDNF gene is target of known DNA regulatory mechanisms but details of its activity-dependent regulation are not fully characterized. We performed a comprehensive analysis of the epigenetic regulation of the turtle BDNF gene (tBDNF) during a neural correlate of associative learning using an in vitro model of eye blink classical conditioning. Shortly after conditioning onset, the results from ChIP-qPCR show conditioning-dependent increases in methyl-CpG-binding protein 2 (MeCP2) and repressor basic helix-loop-helix binding protein 2 (BHLHB2) binding to tBDNF promoter II that corresponds with transcriptional repression. In contrast, enhanced binding of ten-eleven translocation protein 1 (Tet1), extracellular signal-regulated kinase 1/2 (ERK1/2), and cAMP response element-binding protein (CREB) to promoter III corresponds with transcriptional activation. These actions are accompanied by rapid modifications in histone methylation and phosphorylation status of RNA polymerase II (RNAP II). Significantly, these remarkably coordinated changes in epigenetic factors for two alternatively regulated tBDNF promoters during conditioning are controlled by Tet1 and ERK1/2. Our findings indicate that Tet1 and ERK1/2 are critical partners that, through complementary functions, control learning-dependent tBDNF promoter accessibility required for rapid transcription and acquisition of classical conditioning.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factor Neurotrófico Derivado del Encéfalo/genética , Metilación de ADN/genética , Aprendizaje , Proteína Quinasa 3 Activada por Mitógenos/genética , Animales , Cromatina/genética , Proteínas de Unión al ADN/genética , Proteína 2 de Unión a Metil-CpG , Neuronas/metabolismo , Regiones Promotoras Genéticas , Tortugas/genéticaRESUMEN
How the neural substrates for detection of paired stimuli are distinct from unpaired stimuli is poorly understood and a fundamental question for understanding the signalling mechanisms for coincidence detection during associative learning. To address this question, we used a neural correlate of eyeblink classical conditioning in an isolated brainstem from the turtle, in which the cranial nerves are directly stimulated in place of using a tone or airpuff. A bidirectional response is activated in <5 min of training, in which phosphorylated 3-phosphoinositide-dependent kinase-1 (p-PDK1) is increased in response to paired and decreased in response to unpaired nerve stimulation and is mediated by the opposing actions of neurotrophin receptors TrkB and p75(NTR) . Surprisingly, blockade of adenosine 2A (A2A ) receptors inhibits both of these responses. Pairing also induces substantially increased surface expression of TrkB that is inhibited by Src family tyrosine kinase and A2A receptor antagonists. Finally, the acquisition of conditioning is blocked by a PDK1 inhibitor. The unique action of A2A receptors to function directly as G proteins and in receptor transactivation to control distinct TrkB and p75(NTR) signalling pathways allows for convergent activation of PDK1 and protein kinase A during paired stimulation to initiate classical conditioning.
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Proteínas Quinasas Dependientes de 3-Fosfoinosítido/fisiología , Condicionamiento Clásico/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Receptor de Factor de Crecimiento Nervioso/fisiología , Receptor trkB/fisiología , Animales , Tronco Encefálico/fisiología , Nervios Craneales/fisiología , Receptor de Adenosina A2A , Tortugas/fisiologíaRESUMEN
Multiple signaling pathways are involved in AMPAR trafficking to synapses during synaptic plasticity and learning. The mechanisms for how these pathways are coordinated in parallel but maintain their functional specificity involves subcellular compartmentalization of kinase function by scaffolding proteins, but how this is accomplished is not well understood. Here, we focused on characterizing the molecular machinery that functions in the sequential synaptic delivery of GluA1- and GluA4-containing AMPARs using an in vitro model of eyeblink classical conditioning. We show that conditioning induces the interaction of selective protein complexes with the key structural protein SAP97, which tightly regulates the synaptic delivery of GluA1 and GluA4 AMPAR subunits. The results demonstrate that in the early stages of conditioning the initial activation of PKA stimulates the formation of a SAP97-AKAP/PKA-GluA1 protein complex leading to synaptic delivery of GluA1-containing AMPARs through a SAP97-PSD95 interaction. This is followed shortly thereafter by generation of a SAP97-KSR1/PKC-GluA4 complex for GluA4 AMPAR subunit delivery again through a SAP97-PSD95 interaction. These data suggest that SAP97 forms the molecular backbone of a protein scaffold critical for delivery of AMPARs to the PSD during conditioning. Together, the findings reveal a cooperative interaction of multiple scaffolding proteins for appropriately timed delivery of subunit-specific AMPARs to synapses and support a sequential two-stage model of AMPAR synaptic delivery during classical conditioning.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Condicionamiento Clásico , Guanilato-Quinasas/metabolismo , Receptores AMPA/metabolismo , Receptores de Glutamato/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/fisiología , Animales , Conducta Animal , Biotinilación , Membrana Celular/metabolismo , Aprendizaje , Microscopía Confocal , Plasticidad Neuronal , Péptidos/metabolismo , Unión Proteica , Transducción de Señal , Transmisión Sináptica , TortugasRESUMEN
Brain-derived neurotrophic factor (BDNF) is an important regulator of neuronal development and synaptic function. The BDNF gene undergoes significant activity-dependent regulation during learning. Here, we identified the BDNF promoter regions, transcription start sites, and potential regulatory sequences for BDNF exons I-III that may contribute to activity-dependent gene and protein expression in the pond turtle Trachemys scripta elegans (tBDNF). By using transfection of BDNF promoter/luciferase plasmid constructs into human neuroblastoma SHSY5Y cells and mouse embryonic fibroblast NIH3T3 cells, we identified the basal regulatory activity of promoter sequences located upstream of each tBDNF exon, designated as pBDNFI-III. Further, through chromatin immunoprecipitation (ChIP) assays, we detected CREB binding directly to exon I and exon III promoters, while BHLHB2, but not CREB, binds within the exon II promoter. Elucidation of the promoter regions and regulatory protein binding sites in the tBDNF gene is essential for understanding the regulatory mechanisms that control tBDNF gene expression.
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Factor Neurotrófico Derivado del Encéfalo/genética , Genoma , Regiones Promotoras Genéticas , Proteínas de Reptiles/genética , Tortugas/genética , Animales , Secuencia de Bases , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Línea Celular Tumoral , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Exones , Humanos , Ratones , Datos de Secuencia Molecular , Células 3T3 NIH , Proteínas de Reptiles/metabolismoRESUMEN
Brain-derived neurotrophic factor (BDNF) has a diverse functional role and complex pattern of gene expression. Alternative splicing of mRNA transcripts leads to further diversity of mRNAs and protein isoforms. Here, we describe the regulation of BDNF mRNA transcripts in an in vitro model of eyeblink classical conditioning and a unique transcript that forms a functionally distinct truncated BDNF protein isoform. Nine different mRNA transcripts from the BDNF gene of the pond turtle Trachemys scripta elegans (tBDNF) are selectively regulated during classical conditioning: exon I mRNA transcripts show no change, exon II transcripts are downregulated, while exon III transcripts are upregulated. One unique transcript that codes from exon II, tBDNF2a, contains a 40 base pair deletion in the protein coding exon that generates a truncated tBDNF protein. The truncated transcript and protein are expressed in the naïve untrained state and are fully repressed during conditioning when full-length mature tBDNF is expressed, thereby having an alternate pattern of expression in conditioning. Truncated BDNF is not restricted to turtles as a truncated mRNA splice variant has been described for the human BDNF gene. Further studies are required to determine the ubiquity of truncated BDNF alternative splice variants across species and the mechanisms of regulation and function of this newly recognized BDNF protein.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Proteínas de Reptiles/genética , Proteínas de Reptiles/metabolismo , Eliminación de Secuencia , Tortugas , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Factor Neurotrófico Derivado del Encéfalo/química , Regulación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Neuronas/citología , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , Proteínas de Reptiles/química , Transducción de SeñalRESUMEN
Previously, we proposed a two-stage model for an in vitro neural correlate of eyeblink classical conditioning involving the initial synaptic incorporation of glutamate receptor A1 (GluA1)-containing α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid type receptors (AMPARs) followed by delivery of GluA4-containing AMPARs that support acquisition of conditioned responses. To test specific elements of our model for conditioning, selective knockdown of GluA4 AMPAR subunits was used using small-interfering RNAs (siRNAs). Recently, we sequenced and characterized the GluA4 subunit and its splice variants from pond turtles, Trachemys scripta elegans (tGluA4). Analysis of the relative abundance of mRNA expression by real-time RT-PCR showed that the flip/flop variants of tGluA4, tGluA4c, and a novel truncated variant tGluA4trc1 are major isoforms in the turtle brain. Here, transfection of in vitro brain stem preparations with anti-tGluA4 siRNA suppressed conditioning, tGluA4 mRNA and protein expression, and synaptic delivery of tGluA4-containing AMPARs but not tGluA1 subunits. Significantly, transfection of abducens motor neurons by nerve injections of tGluA4 flop rescue plasmid prior to anti-tGluA4 siRNA application restored conditioning and synaptic incorporation of tGluA4-containing AMPARs. In contrast, treatment with rescue plasmids for tGluA4 flip or tGluA4trc1 failed to rescue conditioning. Finally, treatment with a siRNA directed against GluA1 subunits inhibited conditioning and synaptic delivery of tGluA1-containing AMPARs and importantly, those containing tGluA4. These data strongly support our two-stage model of conditioning and our hypothesis that synaptic incorporation of tGluA4-containing AMPARs underlies the acquisition of in vitro classical conditioning. Furthermore, they suggest that tGluA4 flop may have a critical role in conditioning mechanisms compared with the other tGluA4 splice variants.