Добірка наукової літератури з теми "Presynaptic kainate receptors"

Recent reports suggest that kainate acting at presynaptic receptors reduces the release of the inhibitory transmitter GABA from hippocampal neurons. In contrast, in the hypothalamus in the presence of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartate (NMDA) receptor antagonists [1-(4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466) and d,l-2-amino-5-phosphonopentanoic acid (AP5)], kainate increased GABA release. In the presence of tetrodotoxin, the frequency, but not the amplitude, of GABA-mediated miniature inhibitory postsynaptic currents (IPSCs) was enhanced by kainate, consistent with a presynaptic site of action. Postsynaptic activation of kainate receptors on cell bodies/dendrites was also found. In contrast to the hippocampus where kainate increases excitability by reducing GABA release, in the hypothalamus where a much higher number of GABAergic cells exist, kainate-mediated activation of transmitter release from inhibitory neurons may reduce the level of neuronal activity in the postsynaptic cell.

Schaffer collateral synapses in hippocampus show target-cell specific short-term plasticity. Using GFP-expressing Inhibitory Neuron (GIN) transgenic mice that express enhanced green fluorescent protein (EGFP) in a subset of somatostatin-containing interneurons (SOM interneurons), we previously showed that Schaffer collateral synapses onto SOM interneurons in stratum (S.) radiatum have unusually large (up to 6-fold) paired-pulse facilitation. This results from a low initial release probability and the enhancement of facilitation by synaptic activation of presynaptic kainate receptors. Here we further investigate the properties of these kainate receptors and examine their effects on short-term facilitation during physiologically derived stimulation patterns, using excitatory postsynaptic currents recorded in S. radiatum interneurons during Schaffer collateral stimulation in acute slices from juvenile GIN mice. We find that GluR5 and GluR6 antagonists decrease short-term facilitation at Schaffer collateral synapses onto SOM interneurons with no additive effects, suggesting that the presynaptic kainate receptors are heteromers containing both GluR5 and GluR6 subunits. The calcium-permeable receptor antagonist 1-napthyl acetyl spermine (NASPM) both mimics and occludes the effect of the kainate receptor antagonists, indicating that the presynaptic kainate receptors are calcium permeable. Furthermore, Schaffer collateral synapses onto SOM interneurons show up to 11-fold short-term facilitation during physiologically derived stimulus patterns, in contrast to other interneurons that have less than 1.5-fold facilitation. Blocking the kainate receptors reduces facilitation in SOM interneurons by ∼50% during the physiologically derived patterns and reduces the dynamic range. Activation of calcium-permeable kainate receptors containing GluR5/GluR6 causes a dramatic increase in short-term facilitation during physiologically derived stimulus patterns, a mechanism likely to be important in regulating the strength of Schaffer collateral synapses onto SOM interneurons in vivo.

There is intense interest in understanding the molecular mechanisms involved in long-term potentiation (LTP) in the hippocampus. Significant progress in our understanding of LTP has followed from studies of glutamate receptors, of which there are four main subtypes ( α -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA), N -methyl-D-aspartate (NMDA), mGlu and kainate). This article summarizes the evidence that the kainate subtype of glutamate receptor is an important trigger for the induction of LTP at mossy fibre synapses in the CA3 region of the hippocampus. The pharmacology of the first selective kainate receptor antagonists, in particular the GLU K5 subunit selective antagonist LY382884, is described. LY382884 selectively blocks the induction of mossy fibre LTP, in response to a variety of different high-frequency stimulation protocols. This antagonist also inhibits the pronounced synaptic facilitation of mossy fibre transmission that occurs during high-frequency stimulation. These effects are attributed to the presence of presynaptic GLU K5 -subunit-containing kainate receptors at mossy fibre synapses. Differences in kainate receptor-dependent synaptic facilitation of AMPA and NMDA receptor-mediated synaptic transmission are described. These data are discussed in the context of earlier reports that glutamate receptors are not involved in mossy fibre LTP and more recent experiments using kainate receptor knockout mice, that argue for the involvement of GLU K6 but not GLU K5 kainate receptor subunits. We conclude that activation of presynaptic GLU K5 -containing kainate receptors is an important trigger for the induction of mossy fibre LTP in the hippocampus.

The nucleus accumbens, a brain region involved in motivation, attention, and reward, receives substantial glutamatergic innervation from many limbic structures. This excitatory glutamatergic input plays an integral role in both normal and pathophysiological states. Despite the importance of glutamatergic transmission in the nucleus accumbens, the specific receptor subtypes that mediate glutamatergic signaling in this brain region have not been fully characterized. The current study sought to examine the possible role of the kainate subclass of glutamate receptor in the nucleus accumbens. Kainate receptors are relatively poorly understood members of the ionotropic glutamate receptor family and are highly expressed in the nucleus accumbens. Recent studies have highlighted a number of novel pre- and postsynaptic functions of kainate receptors in several other brain regions. Using the whole cell patch-clamp technique, we report the first demonstration of functional kainate receptors on neurons within the core region of the nucleus accumbens. In addition, we present evidence that activation of kainate receptors in this brain region inhibits excitatory synaptic transmission via a presynaptic mechanism.

Visual information is encoded at the photoreceptor synapse by modulation of the tonic release of glutamate from one or more electron-dense ribbons. This release is highest in the dark, when photoreceptors are depolarized, and decreases in grades when photoreceptors hyperpolarize with increasing light. Functional diversity between neurons postsynaptic at the synaptic ribbon arises in part from differential expression of both metabotropic (G-protein-gated) and ionotropic (ligand-gated) glutamate receptor. In the brain, different subunits also modulate the presynaptic active zone. In hippocampus, ionotropic kainate receptors localize to the presynaptic membrane of glutamatergic axon terminals and facilitate depolarization of the synapse (e.g. Lauri et al., 2001). Such facilitation may be helpful in the retina, where consistent depolarization of the photoreceptor axon terminal is necessary to maintain glutamate release in the dark. We investigated whether such a mechanism could be present in primate retina by using electron microscopy to examine the localization of the kainate subunits GluR6/7 at the rod axon terminal, where only a single ribbon synapse mediates glutamate release. We scored 54 rod axon terminals whose postsynaptic space contained one or more GluR6/7-labeled processes and traced these processes through serial sections to determine their identity. Of 68 labeled processes, 63% originated from narrow “fingers” of cytoplasm extending from the presynaptic axon terminal into the postsynaptic cleft. Each rod terminal typically inserts 4–6 presynaptic fingers, and we scored several instances where multiple fingers contained label. Such consistency suggests that each presynaptic finger expresses GluR6/7. The physiological properties of kainate receptors and the geometry of the rod axon terminal suggest that presynaptic GluR6/7 could provide a steady inward current to maintain consistent depolarization of the rod synapse in the long intervals between photons in the dark.

Kainatc receptors (K Rs) arc members of the ionotroplc glutamatc receptor family and are tetramers made up of combinations of GluR5, 6 & 7 and KAI & 2 subunits. KARs arc highly expressed in the brain where they are located both pre- and postsynaptically and regulate network function. Recent work has shown that KARs function was closely related to their subunit composition, This is best described in hippocampus where KARs regulate circuit excitability and where GIuR6 subunit has been implicated in mediating temporal lobe epilepsy. KARs are also highly expressed early in development in sensory pathways and have been shown to be involved in regulating short-term synaptic plasticity during development; however little is known about their subunit specific roles in this mechanism.

Tese de doutoramento em Biologia (Biologia Celular) apresentada à Fac. de Ciências e Tecnologia de Coimbra
Os receptores do glutamato são os principais mediadores da neurotransmissão excitatória no cérebro e também intervêm na sua modulação. Enquanto que a localização e mecanismos de acção de receptores pós-sinápticos do tipo AMPA e NMDA, que suportam a neurotransmissão, são bem conhecidos muito resta a saber acerca da existência, função e mecanismos de acção de receptores que actuam a nível pré-sináptico. A este respeito, muito resta a saber acerca da localização dos receptores de cainato e o seu papel na neurotransmissão. Com o presente trabalho procurámos responder a algumas questões relacionadas com a localização sináptica e função de receptores do glutamato. Na primeira parte do trabalho descrevemos a optimização de uma metodologia bioquímica que permite a obtenção de preparações purificadas de proteínas da zona activa pré-sináptica e da densidade póssináptica. O processo consiste na solubilização sequencial das proteínas não sinápticas em 1% de Triton X-100 a pH 6.0, seguida da solubilização das proteínas pré-sinápticas e sua separação das densidades póssinápticas por aumento do pH para 8.0. Experiências de Western blot usando anticorpos contra proteínas tipicamente pré-sinápticas (SNAP-25 e sintaxina), pós-sinápticas (PSD-95) e não sinápticas (sinaptofisina e NCAM) permitiram verificar a eficiência da separação de proteínas destes compartimentos celulares. De seguida, investigámos a localização subsináptica de diversas subunidades de receptores ionotrópicos e metabotrópicos do glutamato. Observámos que, no caso dos receptores metabotrópicos do glutamato, a subunidade mGluR7 estava localizada maioritariamente na fracção de proteínas da zona activa pré-sináptica. A distribuição subsináptica das outras subunidades estudadas, mGluR1, mGluR2, mGluR4a e mGluR5 foi mais difícil de reconciliar com os resultados de microscopia electrónica existentes na literatura revelando, provavelmente, a limitação do uso da técnica no estudo da localização de receptores que apresentam distribuições particulares, como é o caso de receptores perisinápticos, que não estão localizados nem na zona activa pré-sináptica, nem na densidade pós-sináptica. No caso dos receptores do tipo AMPA, observámos que estes apresentavam uma distribuição subsináptica peculiar, com elevados níveis de imunoreactividade para os anticorpos dirigidos contra as subunidades GluR1, GluR2 e GluR2/3 nas fracções de proteínas da zona activa présináptica, da densidade pós-sináptica e de proteínas não sinápticas. A subunidade GluR4 foi detectada em níveis muito mais modestos e parece predominar pós-sinapticamente. Quanto aos receptores do tipo NMDA, apesar dos vários estudos relatando acções destes receptores ao nível pré-sináptico, detectámos apenas marcação residual para as subunidades NR1, e NR2A-C na zona activa pré-sináptica. A imunoreactividade para todas as subunidades estudadas estava concentrada essencialmente nas densidades póssinápicas e ausente da fracção de proteínas não sinápticas. A pequena amplitude e cinética lenta das correntes sinápticas mediadas por receptores de cainato parecem sugerir uma localização extrasináptica destes receptores, que seriam activados por glutamato difundido para fora da fenda sináptica. No entanto, a manipulação da concentração extracelular de glutamato não altera estas propriedades. Procurámos, portanto, contribuir para o esclarecimento desta aparente discrepância, estudando a localização subsináptica destes receptores. Em estudos funcionais, utilizando sinaptossomas, observámos que a activação de receptores de cainato com baixas concentrações de agonistas aumenta a libertação exocitótica de glutamato tritiado, num processo dependente de Ca2+. Este efeito foi insensível ao antagonista dos receptores AMPA, LY303070 (10 UM), mas foi prevenido pelo antagonista misto para receptores do tipo AMPA e cainato, CNQX (30 UM). Verificámos ainda que a eficiência de modulação da libertação de glutamato por receptores de cainato é superior à conseguida pela simples despolarização da membrana através da elevação da concentração extracelular de KCl apesar do último fenómeno ser mais eficiente em aumentar a [Ca2+]i. Por outro lado, verificámos que o aumento da [Ca2+]i induzido por activação de receptores de cainato (cainato 100 UM) foi só parcialmente inibido pela exposição a bloqueadores de canais de Ca2+ sensíveis à voltagem. Este resultados sugerem fortemente que os receptores pré-sinápticos de cainato estão localizados dentro da zona activa, próximo dos locais de libertação de glutamato sendo, provavelmente, directamente permeáveis a Ca2+. Para comprovar os resultados dos estudos funcionais investigámos a distribuição subsináptica das várias subunidades de receptores de cainato. Estas experiências mostraram que todas as subunidades de receptores de cainato estão localizadas na zona activa pré-sináptica e na densidade póssináptica. A subunidade KA1 mostrou uma localização preferencialmente pós-sináptica. A subunidade GluR7 é uma subunidade dos receptores de cainato cuja função no cérebro é essencialmente desconhecida. A distribuição de mRNA para esta subunidade permite antever uma possível participação em receptores pré-sinápticos nas sinapses das fibras musgosas no hipocampo, pelo que decidimos estudar um possível papel fisiológico de GluR7 ao nível destas sinapses. Através do registo de correntes excitatórias pós-sinápticas, no modo de voltagem imposta, em células piramidais da área CA3 em fatias de cérebro de animais de fenótipo selvagem e animais deficientes para a subunidade GluR7 (GluR7-/-) estudámos uma possível participação desta subunidade em receptores pós-sinápticos de cainato. Observámos que nem a amplitude da resposta dos receptores de cainato nos potenciais excitatórios pós-sinápticos nem a sua cinética estavam alterados em animais GluR7-/-. Assim, sugerimos que esta subunidade não contribui para receptores de cainato a nível póssináptico nas sinapses das fibras musgosas com as células piramidais da área CA3. De seguida, estudámos fenómenos de modulação pré-sináptica através de protocolos de plasticidade de curta e longa duração. Em animais GluR7-/- observámos que a facilitação sináptica devida à aplicação seguida de dois pulsos de estimulação estava significativamente reduzida para intervalos de 10-40 ms entre os pulsos de estimulação, mas apresentava-se normal para intervalos de 100 ms ou superiores, sugerindo uma acção rápida dos receptores de apenas alguns milisegundos. A elevada facilitação observada normalmente nesta sinapse em resposta a um conjunto de 5 estimulações com uma frequência de 20 Hz estava também fortemente reduzida, mostrando que receptores contendo a subunidade GluR7 contribuem para a facilitação sináptica em resposta a estímulos repetidos. Uma outra forma de plasticidade, a facilitação em frequência, que se desenvolve mais lentamente na gama de frequências baixas com estimulação repetitiva, embora não estivesse alterada para frequências mais baixas (0.2 Hz), apresentava-se significativamente reduzida para frequências de estimulação de 0.5 Hz e superiores. A potenciação de longa duração (LTP) observada nas sinapses das fibras musgosas é induzida e expressa a nível pré-sináptico e os receptores pré-sinápticos de cainato, embora inicialmente considerados essenciais para este tipo de plasticidade, desempenham um papel permissivo reduzindo o limiar para a sua indução. Investigámos, por isso, se a subunidade GluR7 teria também um papel preponderante neste tipo de plasticidade sináptica. En animais GluR7-/- a LTP das fibras musgosas estava consideravelmente reduzida, mas não completamente ausente. Adicionalmente, a potenciação pós-tetânica (PTP) estava também severamente reduzida em animais GluR7-/- sem que, no entanto, nenhuma diferença tenha sido observada entre os dois genótipos na potenciação das respostas sinápticas por aplicação de forscolina, indicando que o mecanismo de expressão deste tipo de plasticidade estava intacto. Quer a LTP quer a PTP foram, no entanto, recuperadas para níveis semelhantes aos níveis controlo após elevação da concentração de KCl no meio extracelular ou fornecendo estímulos eléctricos adicionais durante a fase de indução. Embora não tenhamos observado uma facilitação das respostas das sinapses das fibras musgosas pela aplicação de baixas concentrações de cainato (50 nM) a sua inibição foi consistentemente observada em animais de ambos os genótipos pela aplicação de concentrações de cainato superiores a 100 nM. Esta experiência mostrou que a facilitação e inibição das respostas sinápticas pelos receptores de cainato provavelmente não são mediadas pelos mesmos receptores. Mostrámos ainda que não só a subunidade GluR7 tem uma localização sináptica na ausência da subunidade GluR6, e vice versa, mas também que estas duas entidades co-imunoprecipitam em lisados de cérebro, sugerindo a existência de receptores heteroméricos contendo GluR6 e GluR7. Estudos em células HEK transfectadas com GluR6 e GluR7 mostraram que estes receptores heteroméricos são bloqueados pelo antagonista misto de receptores AMPA/cainato, CNQX, e, surpreendentemente, também pelo GYKI 53655, um antagonista considerado selectivo para receptores AMPA. Estabelecemos que estes compostos reduzem a facilitação em frequência em animais controlo mas não em animais GluR7-/-. Adicionalmente, os níveis de facilitação em animais GluR7-/- eram os mesmos observados em animais controlo na presença dos antagonistas, dando um suporte farmacológico aos dados obtidos com a estratégia de delecção genética. Os nossos resultados reforçam o papel dos receptores de cainato como entidades fundamentais no controlo das sinapses glutamatérgicas. A nível pré-sináptico, verificámos que a subunidade GluR7 desempenha um papel fulcral em fenómenos de plasticidade sináptica de curta e longa duração no hipocampo, levantando importantes questões acerca do possível papel deste receptor em outras zonas cerebrais onde a plasticidade sináptica é semelhante à observada nas sinapses das fibras musgosas.
Glutamate receptors play a central role in excitatory neurotransmission in the brain and also in synaptic modulation. Whereas the localization and mechanisms of action of postsynaptic AMPA and NMDA receptors, that support neurotransmission, are more or less well understood, much remains to be studied regarding the existence, function and mechanisms of action of receptors that act at the presynaptic level. With this regard, the synaptic localization of kainate receptors and their role in neurotransmission is one of the most poorly comprehended. With the present effort we sought to answer some of the unresolved issues regarding glutamate receptor localization and function. In the first part of this work we used a new biochemical technique to allow us to obtain purified preparations of proteins from the presynaptic active zone, the postsynaptic density and from non-synaptic pools. This was achieved by the sequential solubilization of non-synaptic proteins in 1% Triton X-100 at pH 6.0, followed by solubilization of presynaptic proteins from the postsynaptic densities by increasing the pH to 8.0. Antibodies directed against typically presynaptic (SNAP-25 and syntaxin), postsynaptic (PSD95) and non-synaptic (synaptophysin and NCAM) proteins allowed us to verify that the methodology yielded preparations of these protein pools with high purity. We next investigated the subsynaptic localization of several subunits of ionotropic and metabotropic glutamate receptors. We found that, for metabotropic glutamate receptors, the mGluR7 subunit was found mainly on the presynaptic active zone, as previously described. The subsynaptic distribution of the other subunits studied, mGluR1, mGluR2, mGluR4a and mGluR5 was more difficult to reconcile with the results from previous immunogold electron microscopy studies, revealing a possible limitation of the solubilization technique in resolving receptors that present particular distributions, such as perisynaptic receptors, that are neither localized in the presynaptic active zone nor in the postsynaptic density. AMPA receptors were found to have a striking subsynaptic distribution, with high amounts of immunoreactivity for GluR1, GluR2 and GluR2/3 in the presynaptic active zone fraction of proteins, in the postsynaptic density and in the non-synaptic pool of proteins. Although there is some evidence that these receptors may be differentially attached to the postsynaptic density, they should not be behaving differently to the solubilization procedure and contribute significantly for the observed presynaptic labbelling. Furthermore, proof for their existence at presynaptic sites is increasingly growing. Despite numerous evidences for actions of NMDA receptors at the presynaptic level, we found only residual labelling for the NR1 and NR2A-C subunits in the pool of proteins from the presynaptic active zone, with the majority of immunoreactivity concentrated at postsynaptic densities. The small labelling of this fraction of proteins for PSD-95 may indicate that labelling at such sites may, in fact, result from slight contamination of the presynaptic active zone faction with proteins from the postsynaptic density. Electrophysiological responses mediated by kainate receptors show small amplitude and slow kinetics that may suggest an extrasynaptic localization and activation by low concentrations of glutamate spilling over from the synaptic cleft. However, manipulating the extracellular glutamate concentration does not change these parameters. Therefore, we sought to add some clarity to this question by investigating the subsynaptic localization of these receptors. In functional studies, using synaptosomes, we observed that activation of kainate receptors with low concentrations of agonists increased the exocytotic release of [3H]glutamate in a Ca2+- dependent manner. This effect was insensitive to the AMPA receptor antagonist, LY303070 (10 UM), but was blocked by the general AMPA/kainate receptor antagonist, CNQX (30 UM). Furthermore, we also observed that kainate (1 UM), although inducing a much more modest increase in the intracellular Ca2+ concentration, was able to significantly modify the release of [3H]glutamate, contrarily to what was observed in a situation of elevated extracellular KCl. These results, together with the fact that the Ca2+ signal was only partially reduced by blockers of voltagesensitive Ca2+ channels, at the supramaximal concentration of 100 UM kainate, suggest that presynaptic kainate receptors are localized close to glutamate release sites, within the active zone, and are probably directly permeable to Ca2+. To look further into the synaptic localization of kainate receptors we performed Western blot experiments in the subsynaptic fractions. This showed that, not only all kainate receptor subunits are localized both in the presynaptic active zone and postsynaptic density but also that they appear to be restricted to these sites of synaptic contact, as shown by the very faint labelling in the non-synaptic pool of proteins. The KA1 subunit revealed to be preferentially localized at the postsynaptic level. Although we showed the subsynaptic localization of kainate receptors and a functional role at the presynaptic level, it is important to understand these parameters at individual synapses and the subunits that are important for synaptic modulation in a more intact system. GluR7 is one subunit of kainate receptors whose function in the brain is unknown. The distribution of mRNA predicts the possibility of its participation to presynaptic kainate receptors at hippocampal mossy fiber synapses and, therefore, we decided to study its possible role at this synapse. By performing whole-cell voltage-clamp recordings from CA3 pyramidal cells in brain slices from wildtype mice and mice lacking GluR7 (GluR7-/-) we first studied the possible contribution of this subunit for postsynaptic receptors. We found that neither receptor kinetics nor the percent contribution of pure kainate receptor-mediated responses to mossy fiber EPSCs were changed in GluR7-/- mice suggesting that, in consistency with anatomical data, GluR7 does not contribute to postsynaptic receptors at mossy fiber-CA3 pyramidal cell synapses. We then turned to presynaptic modulation by using protocols that lead to presynaptic forms of short- and long-term plasticity, which have been shown to be dependent on or modulated by kainate receptors. In animals lacking GluR7 we showed that paired pulse facilitation was significantly impaired at short intervals between stimuli, but normal for intervals of 100 ms or greater, suggesting a fast action of these receptors of only a few milliseconds. The prominent facilitation of mossy fiber responses to a train of 5 stimuli, delivered at a frequency of 20 Hz, was also greatly reduced in GluR7-/- animals, showing that kainate receptors containing this subunit contribute to the facilitation of responses to repetitive stimuli. Frequency facilitation, another form of presynaptic plasticity that develops over a slower time scale with repetitive stimulation in the low frequency range, although not altered at low (0.2 Hz) rates of stimulation, was significantly reduced for stimulation frequencies of 0.5 Hz and higher in the absence of GluR7. Mossy fiber LTP is both induced and expressed presynaptically and presynaptic kainate receptors, although initially thought to be crucial for this process, are now known to have a permissive role by lowering the induction threshold. Therefore, we investigated whether GluR7 had any participation in this form of long-term synaptic plasticity. In animals lacking the GluR7 subunit mossy fiber LTP was strikingly reduced, but not completely absent, when compared to wildtype animals. Furthermore, PTP was also severely impaired in GluR7-/- mice but no difference was found in the forskolininduced potentiation of mossy fiber responses, indicating an intact expression mechanism. Mossy fiber LTP and PTP could, however, be rescued to control levels by either slightly increasing the extracellular KCl concentration or by supplying additional stimuli during induction.