RESUMEN
The presynaptic release apparatus can be specialized to enable specific synaptic functions. Habituation is the diminishing of a physiological response to a frequently repeated stimulus and in Aplysia, habituation to touch is mediated by a decrease in transmitter release from the sensory neurons that respond to touch even after modest rates of action potential firing. This synaptic depression is not common among Aplysia synaptic connections suggesting the presence of a release apparatus specialized for this depression. We found that specific splice forms of ApCaV2, the calcium channel required for transmitter release, are preferentially used in sensory neurons, consistent with a specialized release apparatus. However, we were not able to find a specific ApCaV2 splice uniquely required for synaptic depression. The C-terminus of ApCaV2 alpha1 subunit retains conserved binding to Aplysia rab-3 interacting molecule (ApRIM) and ApRIM-binding protein (ApRBP) and the C-terminus is required for full synaptic expression of ApCaV2. We also identified a splice form of ApRIM that did not interact with the ApCav2 alpha 1 subunit, but it was not preferentially used in sensory neurons.
Asunto(s)
Aplysia , Canales de Calcio , Animales , Canales de Calcio/genética , Canales de Calcio/metabolismo , Aplysia/metabolismo , Células Receptoras Sensoriales/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Potenciales de Acción , Transmisión Sináptica/fisiología , Sinapsis/metabolismo , Calcio/metabolismoRESUMEN
A more thorough description of the changes in synaptic strength underlying synaptic plasticity may be achieved with quantal resolution measurements at individual synaptic sites. Here, we demonstrate that by using a membrane targeted genetic calcium sensor, we can measure quantal synaptic events at the individual synaptic sites of Aplysia sensory neuron to motor neuron synaptic connections. These results show that synaptic strength is not evenly distributed between all contacts in these cultures, but dominated by multiquantal sites of synaptic contact, likely clusters of individual synaptic sites. Surprisingly, most synaptic contacts were not found opposite presynaptic varicosities, but instead at areas of pre- and postsynaptic contact with no visible thickening of membranes. The release probability, quantal size, and quantal content can be measured over days at individual synaptic contacts using this technique. Homosynaptic depression was accompanied by a reduction in release site probability, with no evidence of individual synaptic site silencing over the course of depression. This technique shows promise in being able to address outstanding questions in this system, including determining the synaptic changes that maintain long-term alterations in synaptic strength that underlie memory.
Asunto(s)
Aplysia , Calcio , Animales , Neuronas Motoras , Células Receptoras Sensoriales , Sinapsis , Transmisión SinápticaRESUMEN
Persistent activity of protein kinase M (PKM), the truncated form of protein kinase C (PKC), can maintain long-term changes in synaptic strength in many systems, including the hermaphrodite marine mollusk, Aplysia californica Moreover, different types of long-term facilitation (LTF) in cultured Aplysia sensorimotor synapses rely on the activities of different PKM isoforms in the presynaptic sensory neuron and postsynaptic motor neuron. When the atypical PKM isoform is required, the kidney and brain expressed adaptor protein (KIBRA) is also required. Here, we explore how this isoform specificity is established. We find that PKM overexpression in the motor neuron, but not the sensory neuron, is sufficient to increase synaptic strength and that this activity is not isoform-specific. KIBRA is not the rate-limiting step in facilitation since overexpression of KIBRA is neither sufficient to increase synaptic strength, nor to prolong a form of PKM-dependent intermediate synaptic facilitation. However, the isoform specificity of dominant-negative-PKMs to erase LTF is correlated with isoform-specific competition for stabilization by KIBRA. We identify a new conserved region of KIBRA. Different splice isoforms in this region stabilize different PKMs based on the isoform-specific sequence of an α-helix "handle" in the PKMs. Thus, specific stabilization of distinct PKMs by different isoforms of KIBRA can explain the isoform specificity of PKMs during LTF in AplysiaSIGNIFICANCE STATEMENT Long-lasting changes in synaptic plasticity associated with memory formation are maintained by persistent protein kinases. We have previously shown in the Aplysia sensorimotor model that distinct isoforms of persistently active protein kinase Cs (PKMs) maintain distinct forms of long-lasting synaptic changes, even when both forms are expressed in the same motor neuron. Here, we show that, while the effects of overexpression of PKMs are not isoform-specific, isoform specificity is defined by a "handle" helix in PKMs that confers stabilization by distinct splice forms in a previously undefined domain of the adaptor protein KIBRA. Thus, we define new regions in both KIBRA and PKMs that define the isoform specificity for maintaining synaptic strength in distinct facilitation paradigms.
Asunto(s)
Neuronas Motoras/fisiología , Plasticidad Neuronal , Isoformas de Proteínas/fisiología , Proteína Quinasa C/fisiología , Células Receptoras Sensoriales/fisiología , Animales , Aplysia , Células Cultivadas , Ganglios de Invertebrados/fisiología , Proteínas del Tejido Nervioso/fisiología , Estabilidad ProteicaRESUMEN
There is considerable evidence from both vertebrates and invertebrates that persistently active protein kinases maintain changes in synaptic strength that underlie memory. In the hermaphrodite marine mollusk, Aplysia californica, truncated forms of protein kinase C (PKC) termed protein kinase Ms have been implicated in both intermediate- and long-term facilitation, an increase in synaptic strength between sensory neurons and motor neurons thought to underlie behavioural sensitization in the animal. However, few substrates have been identified as candidates that could mediate this increase in synaptic strength. PKMs have been proposed to maintain synaptic strength through preventing endocytosis of AMPA receptors. Numb is a conserved regulator of endocytosis that is modulated by phosphorylation. We have identified and cloned Aplysia Numb (ApNumb). ApNumb contains three conserved PKC phosphorylation sites and PKMs generated from classical and atypical Aplysia PKCs can phosphorylate ApNumb in vitro and in cells. Over-expression of ApNumb that lacks the conserved PKC phosphorylation sites blocks increases in surface levels of a pHluorin-tagged Aplysia glutamate receptor measured using live imaging after intermediate- or long-term facilitation. Over-expression of this form of ApNumb did not block increases in synaptic strength seen during intermediate-term facilitation, but did block increases in synaptic strength seen during long-term facilitation. There was no effect of over-expression of this form of ApNumb on other putative Numb targets as measured using increases in calcium downstream of neurotrophins or agonists of metabotropic glutamate receptors. These results suggest that in Aplysia neurons, Numb specifically regulates AMPA receptor trafficking and is an attractive candidate for a target of PKMs in long-term maintenance of synaptic strength. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.
Asunto(s)
Proteínas de la Membrana/metabolismo , Plasticidad Neuronal/fisiología , Neuronas/metabolismo , Proteína Quinasa C/metabolismo , Receptores AMPA/metabolismo , Animales , Aplysia , Transporte de Proteínas/fisiologíaRESUMEN
The sensory neuron of Aplysia californica participates in several forms of presynaptic plasticity including homosynaptic depression, heterosynaptic depression, facilitation and the reversal of depression. The calcium channel triggering neurotransmitter release at most synapses is CaV2, consisting of the pore forming α1 subunit (CaV2α1), and auxiliary CaVß, and CaVα2δ subunits. To determine the role of the CaV2 channel in presynaptic plasticity in Aplysia, we cloned Aplysia CaV2α1, CaVß, and CaVα2δ and over-expressed the proteins in Aplysia sensory neurons (SN). We show expression of exogenous CaV2α1 in the neurites of cultured Aplysia SN. One proposed mechanism for heterosynaptic depression in Aplysia is through inhibition of CaV2. Here, we demonstrate that heterosynaptic depression of the CaV2 calcium current is inhibited when a channel with a Y-F mutation at the conserved Src phosphorylation site is expressed, showing the strong conservation of this mechanism over evolution. We also show that the Y-F mutation reduces heterosynaptic inhibition of neurotransmitter release, highlighting the physiological importance of this mechanism for the regulation of synaptic efficacy. These results also demonstrate our ability to replace endogenous CaV2 channels with recombinant channels allowing future examination of the structure function relationship of CaV2 in the regulation of transmitter release in this system.
Asunto(s)
Aplysia , Canales de Calcio/metabolismo , Plasticidad Neuronal , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Células Receptoras Sensoriales/fisiología , Sustitución de Aminoácidos , Animales , Canales de Calcio/genética , Células Cultivadas , Clonación Molecular , Análisis Mutacional de ADN , Motivos EF Hand , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Células Receptoras Sensoriales/enzimología , Tirosina/genéticaRESUMEN
Atypical PKM, a persistently active form of atypical PKC, is proposed to be a molecular memory trace, but there have been few examinations of the role of PKMs generated from other PKCs. We demonstrate that inhibitors used to inhibit PKMs generated from atypical PKCs are also effective inhibitors of other PKMs. In contrast, we demonstrate that dominant-negative PKMs show isoform-specificity. A dominant-negative PKM from the classical PKC Apl I blocks activity-dependent intermediate-term facilitation (a-ITF) when expressed in the sensory neuron, while a dominant-negative PKM from the atypical PKC Apl III does not. Consistent with a specific role for PKM Apl I in activity-dependent facilitation, live imaging FRET-based cleavage assays reveal that activity leads to cleavage of the classical PKC Apl I, but not the atypical PKC Apl III in the sensory neuron varicosities of Aplysia In contrast, massed intermediate facilitation (m-ITF) induced by 10 min of 5HT is sufficient for cleavage of the atypical PKC Apl III in the motor neuron. Interestingly, both cleavage of PKC Apl I in the sensory neuron during a-ITF and cleavage of PKC Apl III in the motor neuron during m-ITF are inhibited by a dominant-negative form of a penta-EF hand containing classical calpain cloned from Aplysia Consistent with a role for PKMs in plasticity, this dominant-negative calpain also blocks both a-ITF when expressed in the sensory neuron and m-ITF when expressed in the motor neuron. This study broadens the role of PKMs in synaptic plasticity in two significant ways: (i) PKMs generated from multiple isoforms of PKC, including classical isoforms, maintain memory traces; (ii) PKMs play roles in the presynaptic neuron.
Asunto(s)
Plasticidad Neuronal/fisiología , Terminales Presinápticos/fisiología , Proteína Quinasa C/metabolismo , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/metabolismo , Animales , Aplysia , Benzofenantridinas/farmacología , Calpaína/farmacología , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Transferencia Resonante de Energía de Fluorescencia , Regulación de la Expresión Génica/efectos de los fármacos , Potenciales de la Membrana/fisiología , Microinyecciones , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/fisiología , Sistema Nervioso/citología , Plasticidad Neuronal/efectos de los fármacos , Cloruro de Potasio/farmacología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Quinasa C/química , Proteína Quinasa C/efectos de los fármacos , Proteína Quinasa C/genética , Serotonina/farmacología , Transducción GenéticaRESUMEN
The inhibition of Aplysia pleural mechanosensory neuron synapses by dopamine and serotonin through activation of endogenous dopaminergic and expressed 5-HT1Apl(a)/b receptors, respectively, involves a reduction in action potential-associated calcium influx. We show that the inhibition of synaptic efficacy is downstream of the readily releasable pool, suggesting that inhibition is at the level of calcium secretion coupling, likely a result of the changes in the calcium current. Indeed, the inhibitory responses directly reduce a CaV2-like calcium current in isolated sensory neurons. The inhibition of the calcium current is voltage independent as it is not affected by a strong depolarizing prepulse, consistent with other invertebrate CaV2 calcium currents. Similar to voltage-independent inhibition of vertebrate nociceptors, inhibition was blocked with Src tyrosine kinase inhibitors. The data suggest a conserved mechanism by which G protein-coupled receptor activation can inhibit the CaV2 calcium current in nociceptive neurons.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Calcio/metabolismo , Dopamina/farmacología , Células Receptoras Sensoriales/fisiología , Serotonina/farmacología , Potenciales Sinápticos/efectos de los fármacos , Animales , Aplysia , Canales de Calcio Tipo R/metabolismo , Células Cultivadas , Inhibidores de Proteínas Quinasas/farmacología , Células Receptoras Sensoriales/efectos de los fármacosRESUMEN
Expression of the 5-HT(1Apl(a)) receptor in Aplysia pleural sensory neurons inhibited 5-HT-mediated translocation of the novel PKC Apl II in sensory neurons and prevented PKC-dependent synaptic facilitation at sensory to motoneuron synapses (Nagakura et al. 2010). We now demonstrate that the ability of inhibitory receptors to block PKC activation is a general feature of inhibitory receptors and is found after expression of the 5-HT(1Apl(b)) receptor and with activation of endogenous dopamine and FMRFamide receptors in sensory neurons. Pleural sensory neurons are heterogeneous for their inhibitory response to endogenous transmitters, with dopamine being the most prevalent, followed by FMRFamide, and only a small number of neurons with inhibitory responses to 5-HT. The inhibitory response is dominant, reduces membrane excitability and synaptic efficacy, and can reverse 5-HT facilitation at both naive and depressed synapses. Indeed, dopamine can reverse PKC translocation during the continued application of 5-HT. Reversal of translocation can also be seen after translocation mediated by an analog of diacylglycerol, suggesting inhibition is not through blockade of diacylglycerol production. The effects of inhibition on PKC translocation can be rescued by phosphatidic acid, consistent with the inhibitory response involving a reduction or block of production of this lipid. However, phosphatidic acid could not recover PKC-dependent synaptic facilitation due to an additional inhibitory effect on the non-L-type calcium flux linked to synaptic transmission. In summary, we find a novel mechanism downstream of inhibitory receptors linked to inhibition of PKC activation in Aplysia sensory neurons.
Asunto(s)
Aplysia/fisiología , Isoenzimas/metabolismo , Inhibición Neural/fisiología , Neurotransmisores/metabolismo , Proteína Quinasa C/metabolismo , Órganos de los Sentidos/fisiología , Células Receptoras Sensoriales/fisiología , Transmisión Sináptica/fisiología , Animales , Células Cultivadas , Activación Enzimática , Potenciales Postsinápticos Excitadores/fisiología , Pleura/inervación , Pleura/fisiologíaRESUMEN
Long-term facilitation (LTF) in Aplysia is a leading model for elucidating the biochemical mechanisms of synaptic plasticity underlying learning. LTF requires translational control downstream of target of rapamycin complex 1. Our lab has previously shown that treatment with the facilitating neurotransmitter, 5-hydroxytryptamine (5-HT), causes a target of rapamycin complex 1-mediated decrease in phosphorylation of eukaryotic elongation factor 2 (eEF2) within the neurites of sensory neurons involved in LTF. Here, we characterize the Aplysia orthologue of eEF2 kinase (eEF2K). We show that the Aplysia eEF2K orthologue contains an S6 kinase phosphorylation site and that a serine-to-alanine mutation at this site blocks the ability of 5-HT to decrease eEF2 phosphorylation in neurites. We also show that within the soma, 5-HT has the opposite effect, decreasing eEF2K phosphorylation at the S6 kinase site and, concomitantly, increasing eEF2 phosphorylation. Surprisingly, while eEF2K over-expression inhibits translation of a marker for internal ribosome entry site-dependent translation, it stimulates the translation of a marker for cap-dependent translation. This study demonstrates that eEF2 is differentially regulated in separate compartments and contributes to a growing body of evidence that inhibition of elongation can stimulate the translation of certain transcripts.
Asunto(s)
Quinasa del Factor 2 de Elongación/biosíntesis , Factor 2 de Elongación Peptídica/biosíntesis , Células Receptoras Sensoriales/metabolismo , Animales , Aplysia , Western Blotting , Proteínas de Caenorhabditis elegans/genética , Células Cultivadas , Clonación Molecular , Quinasa del Factor 2 de Elongación/genética , Regulación de la Expresión Génica/fisiología , Humanos , Inmunohistoquímica , Microscopía Fluorescente , Neuritas/efectos de los fármacos , Factor 2 de Elongación Peptídica/genética , Fosforilación , Biosíntesis de Proteínas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Quinasas S6 Ribosómicas/genética , Proteínas Quinasas S6 Ribosómicas/metabolismo , Serotonina/farmacología , Factores de Transcripción/genéticaRESUMEN
Serotonin (5-hydroxytryptamine, 5HT) is the neurotransmitter that mediates dishabituation in Aplysia. Serotonin mediates this behavioral change through the reversal of synaptic depression in sensory neurons (SNs). However, the 5HT receptors present in SNs and in particular, the receptor important for activation of protein kinase C (PKC) have not been fully identified. Using a recent genome assembly of Aplysia, we identified new receptors from the 5HT(2) , 5HT(4) , and 5HT(7) families. Using RT-PCR from isolated SNs, we found that three 5HT receptors, 5HT(1Apl(a)) , 5HT(2Apl) , and 5HT(7Apl) were expressed in SNs. These receptors were cloned and expressed in a heterologous system. In this system, 5HT(2Apl) could significantly translocate PKC Apl II in response to 5HT and this was blocked by pirenperone, a 5HT(2) receptor antagonist. Surprisingly, pirenperone did not block 5HT-mediated translocation of PKC Apl II in SNs, nor 5HT-mediated reversal of depression. Expression of 5HT(1Apl(a)) in SNs or genistein, an inhibitor of tyrosine kinases inhibited both PKC translocation and reversal of depression. These results suggest a non-canonical mechanism for the translocation of PKC Apl II in SNs.
Asunto(s)
Aplysia/enzimología , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Receptores de Serotonina/fisiología , Animales , Aplysia/genética , Células Cultivadas , Clonación Molecular/métodos , Activación Enzimática/genética , Isoenzimas/fisiología , Filogenia , Proteína Quinasa C/fisiología , Receptores de Serotonina/genéticaRESUMEN
Learning is highly regulated by the pattern of training. In Aplysia, an important organism for the development of cellular and molecular models of learning, spaced versus massed application of the same stimulus leads to different forms of memory. A critical molecular step underlying memory is the serotonin (5HT)-mediated activation of the novel PKC Apl II. Here, we demonstrate that activation of PKC Apl II is highly sensitive to the pattern of 5HT application. Spaced applications downregulate PKC translocation through PKA signaling, whereas massed applications lead to persistent translocation of PKC. Differential regulation of PKC translocation is mediated by competing feedback mechanisms that act through protein synthesis. These studies elucidate a fundamental molecular difference between spaced and massed training protocols.
Asunto(s)
Aplysia/enzimología , Aprendizaje/fisiología , Proteína Quinasa C/metabolismo , Animales , Aplysia/efectos de los fármacos , Células Cultivadas , Aprendizaje/efectos de los fármacos , Memoria/efectos de los fármacos , Memoria/fisiología , Biosíntesis de Proteínas/fisiología , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/enzimología , Serotonina/farmacologíaRESUMEN
The mechanisms by which presynaptic neurones differentially regulate synaptic transmission with multiple postsynaptic targets in the brain are not fully understood. Using intracellular sharp electrode and whole-cell voltage-clamp recordings of soma-soma synapses between identified Lymnaea neurones, we provide direct evidence that quantal size is regulated presynaptically through the coupling of multiple release sites. This coupling effectively multiplies quantal size, thereby providing significant influence over parameters of synaptic transmission that are influenced by quantal size, such as the variance in transmitter release at stationary release probabilities. Variation in the degree of coupling is dependent on the identity of the postsynaptic cell, even though the variation in quantal size is of presynaptic origin. We have therefore demonstrated the presence of a novel mechanism by which presynaptic neurones may differentially regulate quantal size at select synaptic connections, in turn providing them with a means of regulating synaptic transmission with multiple postsynaptic cells.
Asunto(s)
Neurotransmisores/metabolismo , Terminales Presinápticos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Animales , Células Cultivadas , Relación Dosis-Respuesta en la Radiación , Estimulación Eléctrica/métodos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Potenciales Postsinápticos Excitadores/efectos de la radiación , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Modelos Biológicos , Neuronas/citología , Técnicas de Placa-Clamp/métodos , Terminales Presinápticos/efectos de los fármacos , Rianodina/farmacología , Caracoles , Sinapsis/clasificación , Sinapsis/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacosRESUMEN
The Drosophila FMRFamide-related peptide, DPKQDFMRFamide modulates synaptic transmission at the larval neuromuscular junction. The amplitude of excitatory junctional potentials (EJPs) produced by the selective stimulation of motor neuron MN6/7-Ib increases following application of 1 microM DPKQDFMRFamide. EJPs elicited by stimulating motor neuron MNSNb/d-Is, however, exhibit no significant increase with the same concentration of neuropeptide. The mechanisms underlying the modulatory effects of DPKQDFMRFamide were examined using a combination of pharmacological and genetic methods. Three independent lines of evidence implicate CaMKII as an essential effector protein or part of the signal transduction pathway. The effect of the neuropeptide is suppressed by 1 microM KN-93 (CaMKII inhibitor) and by heat-shock induced expression of a CaMKII inhibitor. A heterozygous CaM kinase mutant responds poorly to the peptide.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Drosophila/fisiología , Neuronas Motoras/efectos de los fármacos , Unión Neuromuscular/fisiología , Neuropéptidos/farmacología , Transmisión Sináptica/efectos de los fármacos , Animales , Bencilaminas/farmacología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Estimulación Eléctrica , Larva , Neuronas Motoras/fisiología , Unión Neuromuscular/efectos de los fármacos , Neuropéptidos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Sulfonamidas/farmacología , Transmisión Sináptica/fisiología , Factores de TiempoRESUMEN
The crayfish neuropeptide DRNFLRFamide increases transmitter release from synaptic terminals onto muscle cells. As temperature decreases from 20 to 8 degrees C, the size of excitatory junctional potentials (EJPs) decreases, and the peptide becomes more effective at increasing EJP amplitude. The goal of the present study was to determine whether the enhanced effectiveness of the peptide is strictly a temperature-related effect, or whether it is related to the fact that the EJPs are smaller at low temperature, allowing a greater range for EJP amplitude to increase. Decreasing temperature reduced the number of quanta of transmitter released per nerve impulse (assessed by recording synaptic currents) and increased input resistance in muscle fibers. As in earlier work, the ability of the peptide to increase EJP amplitude was enhanced by decreasing temperature. However, the peptide was also more effective at increasing EJP amplitude when transmitter output was lowered by reducing the ratio of calcium to magnesium ions in the bath. Thus the effectiveness of the peptide may be related to the level of output from the synaptic terminals.