Дисертації з теми "Hippocampal mossy fibers"

In adulthood, mossy fibers (MFs), the axons of granule cells of the dentate gyrus (DG), release glutamate onto CA3 principal cells and interneurons. In contrast, during the first week of postnatal life MFs release -aminobutyric acid (GABA), which, at this early developmental stage exerts a depolarizing and excitatory action on targeted cells. The depolarizing action of GABA opens voltage-dependent calcium channels and NMDA receptors leading to calcium entry and activation of intracellular signaling pathways involved in several developmental processes, thus contributing to the refinement of neuronal connections and to the establishment of adult neuronal circuits. The release of GABA has been shown to be down regulated by several neurotransmitter receptors which would limit the enhanced excitability caused by the excitatory action of GABA. It is worth noting that the immature hippocampus exhibits spontaneous correlated activity, the so called giant depolarizing potentials or GDPs that act as coincident detector signals for enhancing synaptic activity, thus contributing to several developmental processes including synaptogenesis. GDPs render the immature hippocampus more prone to seizures. Here, I explored the molecular mechanisms underlying synaptic transmission and activity-dependent synaptic plasticity processes at immature GABAergic MF-CA3 synapses in wild-type rodents and in mice lacking the prion protein (Prnp0/0 mice). In the first paper, I studied the functional role of kainate receptors (KARs) in regulating GABA release from MF terminals. Presynaptic KARs regulate synaptic transmission in several brain areas and play a central role in modulating glutamate release at adult MF-CA3 synapses. I found that functional presynaptic GluK1 receptors are present on MF terminals where they down regulate GABA release. Thus, application of DNQX or UBP 302, a selective antagonist for GluK1 receptors, strongly increased the amplitude of MF-GABAA-mediated postsynaptic currents (GPSCs). This effect was associated with a decrease in failure rate and increase in PPR, indicating a presynaptic type of action. GluK1 receptors were found to be tonically activated by glutamate present in the extracellular space, since decreasing the extracellular concentration of glutamate with a glutamate scavenger system prevented their activation and mimicked the effects of KAR antagonists. The depressant effect of GluK1 on GABA release was dependent on pertussis toxin (PTx)-sensitive G protein-coupled kainate receptors since it was prevented when hippocampal slices were incubated in the presence of a solution containing PTx. This effect was presynaptic since application of UBP 302 to cells patched with an intracellular solution containing GDP S still potentiated synaptic responses. In addition, the depressant effect of GluK1 on GABA release was prevented by U73122, which selectively inhibits phospholipase C, downstream to G protein activation. Interestingly, U73122, enhanced the probability of GABA release, thus unveiling the ionotropic type of action of kainate receptors. In line with this, we found that GluK1 receptors enhanced MF excitability by directly depolarizing MF terminals via calcium-permeable cation channels. We also explored the possible involvement of GluK1 in spike time-dependent (STD) plasticity and we found that GluK1 dynamically regulate the direction of STD-plasticity, since the pharmacological block of this receptor shifted spike-time dependent potentiation into depression. The mechanisms underlying STD-LTD at immature MF-CA3 synapses have been investigated in detail in the second paper. STD-plasticity is a Hebbian form of learning which consists in bi-directional modifications of synaptic strength according to the temporal order of pre and postsynaptic spiking. Interestingly, we found that at immature mossy fibers (MF)-CA3 synapses, STD-LTD occurs regardless of the temporal order of stimulation (pre versus post or viceversa). However, as already mentioned, while STD-LTD induced by positive pairing (pre before post) could be shifted into STD-LTP after blocking presynaptic GluK1 receptors, STD-LTD induced by negative pairing (post before pre) relied on the activation of CB1 receptors. At P3 but not at P21, endocannabinoids released by the postsynaptic cell during spiking-induced membrane depolarization retrogradely activated CB1 receptors, probably expressed on MF terminals and persistently depressed GABA release in the rat hippocampus. Thus, bath application of selective CB1 receptor antagonists prevented STD-LTD. Pharmacological tools allow identifying anandamide as the endogenous ligand responsible of activity-dependent depressant effect. To further assess whether STD-LTD is dependent on the activation of CB1 receptors, similar experiments were performed on WT-littermates and CB1-KO mice. While in WT mice the pairing protocol produced a persistent depression of MF-GPSCs as in rats, in CB1-KO mice failed to induce LTD. Consistent with these data, in situ hybridization experiments revealed detectable levels of CB1 mRNA in the granule cell layer of P3 but not of P21mice. These experiments strongly suggest that at immature MF-CA3 synapses STD-LTD is mediated by CB1 receptors, probably transiently expressed, during a critical time window, on MF terminals. In the third paper, I studied synaptic transmission and activity dependent synaptic plasticity at immature MF-CA3 synapses in mice devoid of the prion protein (Prnp0/0). The prion protein (PrPC) is a conserved glycoprotein widely expressed in the brain and involved in several neuronal processes including neurotransmission. If converted to a conformationally altered form, PrPSc can cause neurodegenerative diseases, such as Creutzfeldt-Jakob disease in humans. Previous studies aimed at characterizing Prnp0/0 mice have revealed only mild behavioral changes, including an impaired spatial learning, accompanied by electrophysiological and biochemical alterations. Interestingly, PrPC is developmentally regulated and in the hippocampus its expression parallels the maturation of MF. Here, we tested the hypothesis that at immature (P3-P7) MF-CA3 synapses, PrPC interferes with synaptic plasticity processes. To this aim, the rising phase of Giant Depolarizing Potentials (GDPs), a hallmark of developmental networks, was used to stimulate granule cells in the dentate gyrus in such a way that GDPs were coincident with afferent inputs. In WT animals, the pairing procedure induced a persistent increase in amplitude of MF-GPSCs. In contrast, in Prnp0/0 mice, the same protocol produced a long-term depression (LTD). LTP was postsynaptic in origin and required the activation of cAMP-dependent PKA signaling while LTD was presynaptic and was reliant on G protein-coupled GluK1 receptor and protein lipase C downstream to G protein activation. In addition, at emerging CA3-CA1 synapses of PrPC-deficient mice, stimulation of Schaffer collateral failed to induce LTP, known to be PKA-dependent. Finally, we also found that LTD in Prnp0/0 mice was mediated by GluK1 receptors, since UBP 302 blocked its induction. These data suggest that in the immature hippocampus PrPC controls the direction of synaptic plasticity.

Synapsen sind die spezialisierten subzellulären Kontaktstellen im Gehirn, die die Kommunikation zwischen einzelnen Nervenzellen, den Neuronen, auf elektrischem oder chemischem Weg ermöglichen. Anatomisch und physiologisch sind Synapsen jedoch erstaunlich divers, unter anderem abhängig von der untersuchten Hirnregion, der Identität der prä- und postsynaptischen Neurone, den präsynaptisch ausgeschütteten Neurotransmittern und postsynaptischen Rezeptorsystemen. Generell kann die Effektivität oder Stärke synaptischer Übertragung durch unterschiedliche Mechanismen beeinflusst werden. Hier werden nun Mechanismen, Ausprägung und funktionelle Relevanz von Neuromodulation, Kurzzeit- und Langzeit-Plastizität der Stärke der synaptischen Übertragung an der hippokampalen Moosfaser-Synapse erarbeitet. Die vorgestellten Daten konnten mit Hilfe von in vitro experimentellen Ansätzen an der hippokampalen Formation von Mäusen gewonnen werden und durch Analysen und Simulationen aus dem Bereich der theoretischen Biologie bestätigt und erweitert werden.
Chemical synapses are key elements for the communication between nerve cells. This communication can be regulated on various time scales and through different mechanisms affecting synaptic transmission. Amongst these are slow and long-lasting adjustments by endogenous neuromodulators, instantaneous and reversible activity-dependent regulation by short-term plasticity and persistent activity-dependent changes by long-term plasticity. Within this thesis, we have investigated several aspects of modulation of synaptic transmission and its functional relevance at the example of the hippocampal mossy fiber synapse. The presented results were acquired through electrophysiological and microfluorometric experiments at the hippocampal formation of mice and could be verified and substantiated through theoretical analyses, simulations and computational modelling.

Hippocampal mossy fibres (MFs) have been studied intensely in vitro. While many properties of the MFs are well characterised, other findings are debated, particularly in relation to MF long-term potentiation (MF-L TP). MF-L TP is widely accepted as being expressed presynaptically in vitro; however, the induction mechanisms remain unclear. Although kainate receptors (KARs) are generally considered to have a role in MF-L TP induction, the identity of the subtype is debated. Furthermore, the contribution of metabotropic glutamate receptors (mGlu receptors) in this form of plasticity is also controversial. The MFs also exhibit unusual short-term plasticity, including frequency facilitation (FF) which is observed to variable extents in vitro and in conscious rats. An aim of this study was to circumvent the methodological variations, which can affect experimental outcomes in vitro. This was achieved by assessing MF synaptic plasticity in vivo, in anaesthetised rats. A slow-onset MF-L TP was reliably induced and saturated by one train of tetanisation at 100 Hz. MF field excitatory postsynaptic potentials (fEPSPs) were depressed by a group II mGlu receptor agonist, DCG-IV. MF-L TP was also shown to be inducible independently of NMDAR activation. The slow-onset profile of MF-L TP was further investigated and found to be unaffected by altering anaesthetic and tetanisation parameters. Depression, as opposed to facilitation, of the MF fEPSPs occurred in anaesthetised rats during increased frequencies of stimulation. However, facilitation of the fEPSPs was noted in the hippocampus, most likely at the associational/commissural pathway following contralateral or ipsilateral stimulation. MF-L TP persisted in the presence of KAR antagonists or mGlu receptor antagonists. However, MF-L TP was abolished by intrahippocampal co-injection of a KAR antagonist with specific mGlu receptor antagonists. Use of group I mGlu receptor antagonists indicated roles for both mGlu1 and mGlu5 receptors. This study suggests that KARs and mGlu receptors play interchangeable roles in the induction of MF-LTP in vivo.

Dentate microcircuitry is thought to be involved in filtering, integrating, and relaying extrinsic hippocampal inputs to the hippocampus proper, which contributes to memory formation and retrieval. The axons of granule cells are called mossy fibres (MFs), and contain multiple terminal types that form characteristic synaptic connections with their postsynaptic targets. This diversity of presynaptic release sites that exists on the same MF provides an extremely interesting axonal type to study the organizing principles of presynaptic release regulation. A remarkable set of neurotransmitters and receptors present at the MF synaptic complex allow diverse computational modification of information from the dentate gyrus to the hippocampus. There are several types of glutamate receptors expressed at MF, such as group II/III mGluRs and kainate receptors (KARs). Presynaptic KARs modulate transmission at MF-CA3 pyramidal cell synapses; however, it is not known whether presynaptic KARs affect other synapses made by MFs. The aim of the first part of this thesis was to establish the principles of synapse-specific actions of presynaptic KARs in MFs. Combining electrophysiology and calcium imaging, this study provides compelling evidence that presynaptic KARs and Ca²⁺ stores can be activated by glutamate release from a single action potential in a single MF axon. This contributes to short-term, use-dependent facilitation of presynaptic Ca²⁺ entry and glutamate release exclusively at MF-CA3 pyramidal cell synaps, but not at other MF synapses, on hilar mossy cells or interneurons. Thus, our findings indicate that the presynaptic KARs, coupled with intracellular stores, exist in a synapse-specific autoreceptor mechanism. Activation of KARs strengthened MF-CA3 pyramidal cell synapses by increasing the Ca²⁺ influx at giant boutons, which might also contribute to the KAR-dependent hyper-excitability of the MF circuitry related to the mechanisms of temporal lobe epilepsy (TLE). This makes KARs good potential targets for therapies in CNS disorders such as epilepsy and other neurological and psychiatric disorders. The second part of this thesis was to explore the actions on the hippocampus of purified antibodies from a limbic encephalitis (LE) patient. LE is a CNS disease characterized by subacute onset of memory loss and temporal lobe seizures. The serum of these patients strongly labels MFs apparently co-localizing with the VGKC. The patients improve with immunotherapies that reduce the VGKC antibody levels in the serum, thus, strongly suggesting that these antibodies cause the condition. We found that LE serum IgGs enhance CA3 pyramidal cell excitability by blocking α-DTX sensitive VGKCs, which results in the increased release of glutamate. This, in turn, strengthens and desynchronizes MF and CA3 pyramidal cells synaptic transmission. However, these effects were occluded by α-DTX, a Kv1.1, Kv1.2 and Kv1.6 antagonist which, when applied alone, mimicked the action of the LE IgG, suggesting that they may share similar mechanisms of action. In contrast serum taken from healthy control patients had no significant effect under same recording conditions. Thus, this study provides the first evidence that the LE IgG functionally affects VGKC containing Kv1.1, Kv1.2 and/or Kv1.6 at both presynaptic MF axon terminals as well as the postsynaptic somatodendritic domain of CA3 pyramidal cells. Whatever defines the exact nature of LE IgG action, our results suggest that drugs acting specifically as openers of VGKC might help to protect the hippocampus from immune-mediated damage. In conclusion my data is consistent with the increasingly documented idea that MFs play a critical role in regulating the excitability of the hippocampal circuits and the dysfunction of MF transmission profoundly impairs hippocampal function.

L'hippocampe est une région du cerveau située dans le lobe temporal médian. Avec d'autres structures limbiques, l'hippocampe est impliqué dans des processus d'apprentissage et de mémorisation et possède un rôle crucial dans le traitement spatial de l'information. Les synapses de l'hippocampe formées entre les fibres moussues (fm) originaires du gyrus denté et les neurones pyramidaux de CA3 ont reçu une attention particulière, compte tenu de la position stratégique occupée par le gyrus denté à l'entrée de l'hippocampe. En outre les synapses fm- CA3 sont distinctes de la plupart des autres synapses excitatrices du système nerveux central par leurs propriétés morphologiques et physiologiques uniques. Cela soulève la question de savoir si ces propriétés uniques reflètent aussi un rôle fonctionnel unique dans le traitement de l'information effectué par cette synapse au sein du microcircuit de l'hippocampe. Malheureusement nous ne savons que peu de choses sur la façon dont les cellules granulaires modulent l'activité des neurones de CA3 dans le réseau intact in vivo (Henze et al, 2002 ; Hagena et Manahan - Vaughan, 2010, 2011). Le manque d'information est dû au fait que la manipulation classique des circuits neuronaux par des approches électriques, pharmacologiques et génétiques manque de précisions spatiale et temporelle in vivo. L'utilisation de la stimulation extracellulaire de fibres moussues peut conduire à l'activation polysynaptique de cellules pyramidales de CA3, qui peuvent ensuite contaminer les réponses enregistrées. Par ailleurs, l'utilisation de critères trop conservateurs peut conduire à l'exclusion des réponses provenant des fibres moussues «purs» aux propriétés méconnues (Henze et al., 2000). Toutefois, le développement récent et rapide de l’optogénétique dans les neurosciences a fourni de nouveaux outils offrant une sélectivité spatiale élevée (activation optique spécifique de la cellule), et une grande précision temporelle (à l'échelle de la milliseconde), permettant la dissection et l'étude des circuits neuronaux in vivo. L'objectif de ma thèse était de mieux comprendre les mécanismes et les conséquences physiologiques de la plasticité synaptique à court terme se produisant à la synapse formée entre les fibres moussues et les neurones pyramidaux de CA3 dans le cerveau de souris intact. La présente thèse se compose de deux parties principales. Dans la première partie, j'ai exploré de nouveaux outils optogénétiques dans le but de contrôler l'activité des cellules granulaires à l’aide d’impulsions de lumière. La stimulation optogénétique repose sur l'activation du canal ionique channelrhodopsin - 2 - lumière fermée ( ChR2 ) par une lumière bleue et induit des potentiels d'action sur une large gamme de fréquences de stimulation. J'ai aussi observé que la stimulation optique peut être utilisée pour déclencher la plasticité à court terme au niveau des synapses fm-CA3.Dans la deuxième partie j'ai affiné la méthodologie de stimulation optogénétique in vivo pour la caractérisation non invasive du fonctionnement synaptique des synapses fm- CA3. La fiabilité de la stimulation optogénétique d'une population neuronale génétiquement ciblée ainsi que la résolution d'une seule cellule obtenue en utilisant des enregistrements de cellules entières sont des étapes importantes vers une meilleure compréhension du rôle fonctionnel des fibres moussues dans le réseau de l'hippocampe in vivo
The hippocampus is a brain region located in the medial temporal lobe. Along with other limbic structures, the hippocampus is involved in learning and memory processes and has a crucial role in spatial information processing. Within the hippocampus synapses made between mossy fibers (mf) originating from the dentate gyrus and CA3 pyramidal neurons have received particular attention, given the strategic position occupied by the dentate gyrus at the entrance of the hippocampus. Moreover mf-CA3 synapses are distinct from most of other excitatory synapses in the central nervous system for their unusual morphological and physiological properties. This raises the question if these unique properties reflect a unique functional role in information processing carried out by this synapse within the microcircuit of the hippocampus. Unfortunately very little is known on how granule cells modulate the activity of CA3 neurons in the intact network in vivo (Henze et al., 2002; Hagena and Manahan-Vaughan, 2010, 2011). The paucity of information is due to the fact that classical manipulation of neuronal circuits using electrical, pharmacological and genetic approaches lack spatial and temporal precision in vivo. The use of bulk extracellular stimulation may lead to polysynaptic activation of CA3 pyramidal cells, which can subsequently contaminate putative mossy fibers synaptic responses measured in CA3 pyramidal cells. The use of overly conservative criteria on the other side may lead to the exclusion of “pure” mossy fibers responses with unexpected properties (Henze et al., 2000).However the recent and fast growth of optogenetics in neuroscience has provided new tools with high spatial selectivity (cell specific optical activation) and temporal precision (at the millisecond scale), allowing the dissection and investigation of neuronal circuits in vivo. The aim of my thesis was to gain insight into the mechanisms and the physiological consequences of short-term synaptic plasticity occurring at mossy fibers to CA3 pyramidal neurons synapses in the intact mouse brain. The present thesis consists of two main parts. In the first part I explored new optogenetic tools to control the activity of granule cells with pulses of light. Optogenetic stimulation, which relies on the activation of the light-gated ion channel channelrhodopsin-2 (ChR2) by blue light reliably induced action potentials over a wide range of frequencies of stimulation. I also found that optical stimulation can be used to trigger short term plasticity at mf-CA3 synapses. In the second part I refined optogenetic stimulation methodology in vivo for non-invasive characterization of synaptic functioning of the mf-CA3 synapses. The reliability of optogenetic stimulation of a genetically targeted neuronal population together with the single cell resolution obtained using whole-cell recordings are important steps towards a better understanding of the functional role of the mossy fibers in the hippocampal network in vivo

Les synapses fibres moussues de l‘hippocampe entre le gyrus denté et les cellulespyramidales de CA3 sont caractérisées par leur morphologie particulière, et par leurspropriétés distinctives de transmission synaptique et de plasticité présynaptique. Cessynapses sont parfois appelées «détonatrices» pour leur rôle fonctionnel dansl‘encodage de la mémoire épisodique. Cependant, les mécanismes moléculaires à labase des propriétés spécifiques de ces synapses restent peu connus. Ce travail estcomposé de deux parties principales:1) Phénotypage des synapses fibres moussues de l'hippocampe chez les sourisVAMP7 KOVAMP7 est une protéine SNARE vésiculaire de la famille des longins, qui joue unrôle dans la croissance des neurites durant le développement. Dans le cerveauadulte, VAMP7 est enrichi dans un sous-ensemble de terminaisons nerveuses, enparticulier dans les fibres moussues de l‗hippocampe. Nous avons analysé lafonction de VAMP7 dans la libération de neurotransmetteurs par une caractérisationextensive de la transmission synaptique et des mécanismes de plasticité de cettesynapse. L'absence de VAMP7 ne cause pas de graves déficits développementauxou neuronaux (Sato et al., 2011; Danglot et al., 2012). Les mécanismesprésynaptiques de la plasticité à court terme de la fibre moussue de l‘hippocampesemblent également normaux, pour des raisons éventuelles qui seront discutées.2) Circuits du CA3 examinés par traçage viral et enregistrements de pairesNous avons développé une technique pour établir des enregistrements en pairesentre cellules en grain du gyrus denté connectées et cellules pyramidales CA3 (GCCA3),sur des cultures organotypiques de tranches d'hippocampe de souris. Pouridentifier les partenaires présynaptiques directs à une cellule pyramidale CA3 ciblée,nous avons combiné l‘électroporation cellulaire unitaire et le traçage mono-transsynaptiquebasé sur un virus de la rage recombinant et pseudotypé. Nous avonstransfecté une cellule pyramidale CA3 unique par tranche avec les plasmides codantla glycoprotéine d‘enveloppe du virus de la rage (RG), un rapporteur fluorescent, etla protéine TVA (récepteur de surface apparenté au EnvA, qui n'a pas d‘homologuechez les cellules de mammifères). Les tranches ont ensuite été infectées avec levirus de la rage recombinant et pseudotypé. Après 3-4 jours, le traçage mono-transsynaptiquerévèle les entrées présynaptiques de ce neurone unique. Ensuite, nousavons pu établir des enregistrements de paires entre les cellules en grain-CA3connectés, ainsi que de quantifier les partenaires présynaptiques de la cellulepyramidale CA3 de départ
The hippocampal mossy fiber is characterized by its particular morphology, distinctsynaptic transmission and presynaptic plasticity. Moreover, this synapse has beencalled ―teacher‖ or ―detonator‖ for its proposed functional role in episodic memoryencoding. Nevertheless, the molecular mechanisms underlying its specific functionalproperties remain elusive. This work is composed of two main parts:1) Phenotyping Hippocampal Mossy Fiber Synapses in VAMP7 KO MiceVAMP7 is a vesicle SNARE of the longin family important in neurite growth duringdevelopment. In the adult brain, VAMP7 is enriched in a subset of nerve terminals,particularly at the hippocampal mossy fiber. We analyzed VAMP7 function inneurotransmitter release by characterizing basal and evoked transmission at thissynapse in KO mice and fully tested hypotheses relevant to short-term plasticity.Loss of VAMP7 has been previously reported not to cause major developmental orneurological deficits (Sato et al., 2011; Danglot et al., 2012). Presynapticmechanisms of short-term plasticity at the hippocampal mossy fiber also seemunaffected for potential reasons that will be discussed.2) CA3 Circuits Probed with RABV-Tracing and Paired RecordingsWe developed a technique to establish paired recordings between connected dentategyrus granule cells and CA3 pyramidal cells (GC-CA3) in mouse hippocampalorganotypic slice cultures. To identify direct presynaptic partners to a defined targetCA3 pyramidal cell, we combined single-cell electroporation (SCE) and mono-transsynaptictracing based on a pseudotyped, recombinant rabies virus (EnvApseudotyped RABV ΔG). Using SCE we transfected a single CA3 pyramidal cell perslice with the plasmids encoding: the RABV envelope glycoprotein (RG), afluorescent reporter, and TVA (the EnvA cognate surface receptor, which has nohomologue in mammalian cells). The slices were subsequently infected with EnvApseudotyped RABV ΔG. After 3-4 days, the RABV mono-trans-synaptic tracingrevealed the presynaptic inputs of that single neuron. Then, we were able toestablish paired recordings between connected GC-CA3 cells, as well as to quantifythe presynaptic partners of the starter CA3 pyramidal cell
De mosvezel van de hippocampus kenmerkt zich door een bijzondere morfologie,uitzonderlijke synaptische transmissie en presynaptische plasticiteit. De synapswordt ook wel "leraar" of "detonator" genoemd vanwege zijn waarschijnlijke rol in decodering van het episodisch geheugen. Toch blijven de specifieke moleculairemechanismen van dit synaps onbekend. Dit werk bestaat uit twee delen:1) Fenotypering van mosvezel synapsen van de hippocampus in VAMP7 KO muizenVAMP7 is een vesicle-SNARE van de longin familie van belang bij de groei vanneurieten tijdens de ontwikkeling. In de volwassen hersenen, wordt VAMP7 verrijkt ineen subset van zenuwuiteinden, vooral in de mosvezel van de hippocampus. Weanalyseerden VAMP7 functie in neurotransmitter afgifte door het karakteriseren vanbasale en opgeroepen transmissie bij deze synaps in KO muizen. Eerder is algesteld dat gebrek aan VAMP7 niet leidt tot grote ontwikkelings- of neurologischeafwijkingen (Sato et al., 2011; Danglot et al., 2012). Presynaptische mechanismenvan korte termijn plasticiteit in de mosvezel van de hippocampus lijken ookonaangetast te zijn, de mogelijke redenen hiervoor zullen worden besproken.2) CA3 circuits onderzocht met behulp van RABV-tracing en gekoppelde opnamesWe ontwikkelden een techniek om gekoppelde opnames tussen korelcellen van degyrus dentatus en aangesloten CA3 piramidale cellen (KC-CA3) op zogenaamde‗mouse hippocampal organotypic slice cultures‘ te meten. Om rechtstreeksepresynaptische partners te identificeren van een specifieke CA3 piramidale cel,combineerden we single-cell electroporation (SCE) en mono-trans-synaptic tracingop basis van een pseudo-typed, recombinant rabiësvirus (EnvA pseudogetypedRABV ΔG). Met behulp van SCE transfecteerde we één CA3 piramidale cel per slicemet plasmiden die coderen voor: het RABV glycoproteïne-envelop (RG), eenfluorescerende reporter, en TVA (de aan EnvA verwante oppervlakte receptor diegeen homoloog in zoogdiercellen heeft). De slices werden vervolgens geïnfecteerdmet ENVA pseudogetyped RABV ΔG. Na 3-4 dagen bracht de RABV mono-transsynaptischetracing de presynaptische ingangen van die ene neuron aan het licht.Hierna konden we gekoppelde opnames doen tussen verbonden KC-CA3 cellen.Daarnaast konden we de presynaptische partners van de starter CA3 pyramidale celkwantificeren

La maladie d’Alzheimer (MA) est la forme la plus commune de démence chez les personnes âgées. La maladie se caractérise par des pertes de fonctions cognitives et plusieurs études ont montré une étroite relation entre la neuroinflammation, les déficits synaptiques et la perte des fonctions cognitives dans la MA. L'importance de la composante neuroinflammatoire a été démontrée essentiellement grâce à des données épidémiologiques rapportant une prévalence diminuée de 40 à 70% chez des patients traités chroniquement par des anti-inflammatoires non stéroïdiens (AINS) pour d'autres pathologies. Les AINS sont des inhibiteurs des enzymes de synthèse des prostaglandines. Les prostaglandines sont des métabolites de l’acide arachidonique. Parmi ces prostaglandines, la PGE2 est connue pour moduler la transmission et les plasticités synaptiques dans l’hippocampe et son expression est fortement augmentée dans la maladie d’Alzheimer. De nombreux travaux rapportent l'existence de déficits synaptiques dans la MA, notamment dans l'hippocampe, siège de la mémoire et de l’apprentissage. Ces travaux se sont focalisés sur les déficits postsynaptiques à la synapse archétypique formée entre les cellules pyramidales CA3 et CA1. A l'inverse, la synapse formée entre les fibres moussues et les cellules pyramidales CA3 (FM-CA3) exprime des plasticités présynaptiques atypiques, à court et à long terme, indépendantes des récepteurs NMDA et il n'existe que très peu d'études concernant ces plasticités dans le contexte de MA. L’objectif de cette étude a été de montrer l’implication de PGE2 dans les déficits synaptiques à la synapse FM-CA3 dans un modèle murin de la MA, la souris double transgénique APPswe/PS1ΔE9 (APP/PS1). Nos résultats montrent que l’application exogène de PGE2 chez des souris sauvages entraîne un déficit de plasticité uniquement sur la potentialisation à long terme (PLT) exprimée présynaptiquement via l’activation spécifique du récepteur EP3. Nous montrons aussi que dans la souris APP/PS1, seule cette PLT présynaptique est impactée à partir de 12 mois. Enfin, ce déficit de la PLT présynaptique pour la souris APP/PS1 est réversé par un inhibiteur spécifique des récepteurs EP3 montrant ainsi un rôle clé pour la signalisation PGE2 - EP3 dans les déficits synaptiques hippocampaux de ce modèle murin de la maladie d’Alzheimer
Alzheimer’s disease (AD) is the most common form of dementia in elder people characterized by a loss of cognitive function linked to synaptic deficits. There is considerable evidence that neuroinflammation and AD are intimately linked. The key role of neuroinflammation in the course of the disease was figured out by epidemiological studies reporting a reduced prevalence to develop AD for patients chronically treated with Non-Steroidal Anti-Inflammatory Drugs (NSAIDs). Prostaglandins are lipidic mediators derived from arachidonic acid and their synthesis is inhibited by NSAIDs. Among prostaglandins, PGE2 is known to modulate synaptic transmission and plasticity in the hippocampus and its concentration is higher in brains from AD patients. Numerous studies have reported synaptic deficits in the course of AD, mainly in the hippocampus which is essential for cognitive functions like learning or memory formation. The vast majority of these studies were focused on postsynaptic deficits at the canonical CA3-CA1 synapse. On the opposite, the synapse between mossy fiber and CA3 pyramidal cell (Mf-CA3) that express presynaptic short-term and long-term plasticity, was poorly studied in the context of AD. The aim of my project was to decipher the involvement of PGE2 in synaptic deficits in a mouse model of AD, the APPswe/PS1ΔE9 (APP/PS1). Our results show that acute application of PGE2 on wild type young mice impairs only presynaptic long term potentiation (LTP) at the Mf-CA3 synapse via the specific activation of EP3 receptor. In APP/PS1 mice, we demonstrate that the sole deficit at the Mf-CA3 synapse is an impairment of the presynaptic LTP at 12 months of age. Finally we demonstrate that the impaired presynaptic LTP in APP/PS1 mice can be rescued by the acute application of a specific EP3 receptor antagonist, pointing out the key role of PGE2 - EP3 signaling pathway in synaptic deficits in hippocampus in a mouse model of AD

Cette thèse a pour objectif spécifique d’explorer les événements précoces pouvant être à l’origine du bourgeonnement aberrant des fibres moussues (FM) du gyrus denté, une réorganisation majeure dans l’Epilepsie du Lobe Tempora (ELT). Nous avons utilisé le modèle pilocarpine d’ELT chez le rat afin de montrer que la transmission GABAergique jouait un rôle prépondérant dans la formation des FM aberrantes au cours de l’épileptogenèse. Ceci étant due à une altération de l’homéostasie chlore, suite à une augmentation de l’expression du co-transporteur NKCC1 et une diminution du co-transporteur KCC2. Nos résultats ont démontré que le récepteur aux neurotrophines p75NTR était un médiateur de l’action trophique de la réponse GABAergique dépolarisante sur le bourgeonnement aberrant des FM. Le blocage de l’action dépolarisante de la transmission GABAergique via l’utilisation de la bumétanide, a permis de réduire le bourgeonnement aberrant des MF en réduisant l’expression de p75NTR. Enfin, l’application transitoire de la bumétanide au cours de l’épileptogenèse a abouti à la réduction du nombre de crises récurrentes et spontanées au cours de la phase chronique d’ELT chez le rat. Ce travail a permis de dévoiler les mécanismes moléculaires sous-jacents de la réorganisation du réseau neuronal glutamatergique consécutif à une crise inaugurale dans un modèle d’ELT. Dans l'ensemble, cette thèse apporte un éclairage nouveau sur l’importance de l’interaction de la signalisation GABAergique avec les neurotrophines afin d’orchestrer la plasticité réactive au sein de l’hippocampe dans TLE
The present dissertation undertakes to investigate the early triggering events of the mossy fiber sprouting (MFS) in the dentate gyrus, a hallmark of hippocampal reactive plasticity in Temporal Lobe Epilepsy (TLE). We used the rat pilocarpine model of TLE to show that altered GABAA receptor-mediated transmission play a key role in the formation of early ectopic MFS during epileptogenesis. This is likely due to a compromised chloride homeostasis, as a result of increased expression of chloride loader NKCC1 and downregulation of the neuronal chloride extruder KCC2. We next addressed the mechanistic action of depolarizing GABAAR responses with regard to neurotrophin signaling. Our findings uncovered that the pan neurotrophin receptor p75 (p75NTR) mediated the sculpting action of depolarizing GABAAR responses on the ectopic MFS. Blockade of depolarizing GABAAR responses using the loop diuretic bumetanide reduced abnormal p75NTR subsequently decreased the ectopic MFS. Finally, transitory application of bumetanide during epileptogenesis resulted in reduction of spontaneous and recurrent seizures during the chronic phase of TLE. The rationale of this work is that unveiling the molecular mechanisms underlying the hippocampal post-seizure glutamatergic network rewiring will help to drive future novel therapeutic avenues involving chloride homeostasis and neurotrophin interplay. Overall, this dissertation shed a new light on how GABAergic transmission and neurotrophin signaling crosstalk can orchestrate reactive hippocampal plasticity in TLE

La Polarité Cellulaire Planaire (PCP) est une voie de signalisation originellement identifiée chez les invertébrés pour son rôle dans l’établissement d’une asymétrie cellulaire perpendiculaire à l’axe apico‐basal. Elle définit une polarité dans le plan d’un épithélium et coordonne cette polarité dans tout l'épithélium. L'activation de la voie PCP conduit à une réorganisation ducyto squelette en passant par une modulation des zones d'adhésion, régulant ainsi la forme et les mouvements des cellules. La voie de signalisation de la PCP est conservée tout au long de l'évolution jusqu'au mammifères, et contrôle la morphogénèse de divers tissus dont les tissus épithéliaux et mésenchymateux, ainsi que pour les tissues cardiaques, osseux, pulmonaire ou encore rénaux, mais aussi le système nerveux pour n'en citer que quelques‐uns.Afin d'identifier le rôle de vangl2, un des gènes centraux de la PCP, dans la mise en place de la circuiterie hippocampale, nous avons créé un modèle murin où vangl2 est supprimé de façon conditionnelle (cKO) dans le télencéphale à des stades précoces de l’embryogénèse. J’ai d'abord montré que Vangl2 est enrichi dans les neurones immatures de la zone sous granulaire du DG, ainsi que dans l’arborisation des neurites (axones et dendrites) des cellules granulaires (CG) du gyrus denté (DG) de l’hippocampe. Ainsi, Vangl2 est enrichi dans le stratum lucidum (sl), une région dense en contacts synaptiques entre le DG et le CA3. Dans cette région a lieu une synapse très particulière entre l'axone des CG, la fibre moussue (Mf) qui forme des boutons géants (MfB) et les excroissances épineuse (TE) issues de la partie proximale des dendrites apicaux. L'analyse structurale et ultra structurale de ces épines démontre que l'élargissement et la complexification de la synapse MfB/TE est bloquée dans nos mutants, alors que les zones actives (PSD) des épines sont présentes, mais réorganisées. De façon intéressante,dans une zone plus distale des dendrites des neurones du CA3 (sl), les épines sont, elles, plus grosses, suggérant un remodelage complexe du réseau en l'absence de vangl2. Enfin, j’ai pu montrer que ces défauts morphologiques étaient corrélés à des problèmes de mémoire complexe (mémoire déclarative) qui dépendent de l’hippocampe mais aussi du cortex. Cette étude montre pour la première fois l’importance du signal PCP dans maturation in vivo d’un circuit hippocampique spécifique ainsi que ces conséquences cognitives. D'autres résultats in vitro montrent que la suppression de vangl2 augmente la vitesse de déplacement des cônes de croissance sur des substrats de N‐cadhérine. J’ai utilisé la microscopie en super résolution spt‐PALM‐TIRF pour montrer que cette augmentation de croissance est inversement proportionnelle à la vitesse du flux rétrograde d’actine. Des expériences de FRAP permettent de suggérer que les molécules de N‐cadhérine engagées dans des interactions hémophiliques (adhésion) est plus importante dans les mutants vangl2 Je propose que Vangl2 contrôle le recyclage et la stabilité des protéines N‐cadhérine dans les sites d’adhésion afin de réguler localement les dynamiques d’actine et par conséquent la croissance neuronale
Planar Cell Polarity (PCP) is a signaling pathway originally known for its role in the establishment of cellular asymmetry perpendicular to the apico‐basal axis, in the plane of an epithelium. PCPsignaling has been shown to be crucial for many tissue patterning, including epithelial and mesenchymal tissue, but also cardiac, lung, bone, or kidney tissues, to cite a few. PCP signaling controls the regulation of cellular movement via the control of adhesion turnover and cytoskeleton reorganization. Vangl2 is one of the most upstream core PCP proteins that has been implicated in the recent years in various neuronal mechanisms, such as axonal guidance, dendrite morphogenesis or synaptogenesis. However, most of these studies rely on acute downregulation of the gene in vitro or in the use of a mouse presenting a spontaneous mutation of this gene, called Loop‐tail (Vangl2Lp) which causes the death of the embryo at birth. Moreover, the Vangl2Lp form of this protein has been described has a dominant‐negative form, making it difficult to untangle the molecular mechanism leading to the many phenotypes (included neuronal ones) reported inhomozygotes Looptail mice. To bypass this problem we created a conditional knockout (cKO) mouse in which vangl2 is deleted in the telencephalon during early embryogenesis. First, I analyzed the profile of expression of the protein during the first 3 weeks after birth, and I show that Vangl2 is specifically targeted to the arborization of granular cells (GC) of the dentate gyrus (DG) of the hippocampus, and excluded from cell bodies. Also, the protein was highly enriched in immature neurons of the subgranular zone of the DG, and in the stratum lucidum, a region of high‐density contacts between the GC and the CA3. In this region, a special type of synapse is formed: the Mossy Fiber Bouton (MfB) / Thorny Excrescence (TE) synapse. These synapses are bigger and more complex than conventional synapses. I then performed a structural and ultrastructural analysis of the DG/CA3 circuit in the Vangl2 cKO mice in order to understand the role of Vangl2 in the hippocampus maturation. For this, I used stereotaxic mice infection viruses, and Serial block face scanning electron microscopy (SBFsEM) with 3D reconstruction. Results show that in cKO mice, Mfs fasciculation is mildly impacted, and that the enlargement and complexification of the MfB/TE synapse is arrested, with TEs almost absent. I was able to link these morphological abnormalities to deficits in complex hippocampal‐dependent learning tasks. This work demonstrates for the first time the importance of PCP signaling for the in vivo maturation of a specific hippocampal circuit and its specific cognitive consequences. Next, I attempted to identify the functional consequences of vangl2 deletion on young hippocampal neuron maturation. My results confirm that Vangl2 is expressed in young hippocampal neurons and that the deletion of the gene affected neurite outgrowth on Ncadherin substrate. I used spt‐PALM‐TIRF super‐resolution microscopy to show that this increased neurite outgrowth was inversely proportional to a decrease in actin retrograde flowand to a decrease in the number of directed actin trajectories. These results strongly suggest that N‐cadherin adhesions are affected by Vangl2 deletion. FRAP experiments demonstratedthat in Vangl2 cKO neurons the recovery of N‐cadherin molecules engaged in homophilicbindings (adhesion) was decreased, suggesting that the turnover of N‐cadherin involved inadhesion is reduced. Altogether, I propose that Vangl2 controls the turnover/stability of Ncadherin proteins at adhesion sites to regulate local actin dynamics and consequently neuronal outgrowth

Les synapses se forment selon plusieurs étapes comprenant la stabilisation des contacts nouvellement formés et leur maturation. Ces différentes étapes dépendent d’une mise en place coordonnée entre la terminaison pré- et postsynaptique. Les protéines composant la présynapse et les récepteurs ionotropiques du glutamate ont des rôles clés dans ces processus. Lors de ma thèse, je me suis intéressé à l’implication de la protéine présynaptique Bassoon lors de la maturation des synapses glutamatergiques entre les fibres moussues et les cellules pyramidales de CA3 dans l’hippocampe. Cette synapse constitue un modèle attractif pour l’étude de la maturation synaptique car elle suit des étapes de maturation morphologique et fonctionnelle bien définies. Bassoon est une des premières protéines se mettant en place au niveau des contacts synaptiques nouvellement formés. Par des approches électrophysiologiques, nous avons montré que la protéine Bassoon était importante pour l’organisation du site de libération de neurotransmetteur durant les deux premières semaines de vie post-natale chez la souris.Les récepteurs kaïnate jouent un rôle important dans la régulation de l’activité de réseau au cours du développement post-natal. Cependant l’impact de l’activation de ces récepteurs sur la maturation synaptique est peu connu. J’ai pu mettre en évidence un délai dans la maturation fonctionnelle de la synapse fibre moussue/cellule pyramidale de CA3 chez les souris déficientes pour la sous-unité GluK2 des récepteurs kaïnate (GluK2-/-). Afin de comprendre si ce délai de maturation fonctionnelle est corrélé à un retard dans la maturation morphologique de cette synapse, nous avons mis en place des infections de lentivirus codant pour une protéine membranaire fluorescente (YFP) chez le souriceau nouveau-né (P1-P2). A l’aide de microscopie confocale et de reconstruction en 3D, nous avons ainsi pu décrire la maturation morphologique de la synapse fibre moussue/cellule pyramidale de CA3. Cela m’a également permis de corréler la maturation fonctionnelle à la maturation morphologique et mes résultats montrent également un retard dans la mise en place des synapses chez les souris GluK2-/-. L’ensemble de cette étude révèle l’importance de l’activité synaptique et de la coordination entre mise en place de la pré- et de la postsynapse au cours de la maturation synaptique
The formation of synapses follows different steps including synaptogenesis and maturation. These different steps depend on coordinated pre- and post-synaptic assembly. Pre-synaptic proteins and ionotropic glutamate receptors play a central role in these processes. During my thesis, I have been interested in the implication of the presynaptic protein Bassoon in the maturation of the hippocampal mossy fiber to CA3 pyramidal cell glutamatergic synapses. This synapse constitutes an attractive model for the study of synaptic maturation because it follows several steps of defined morphological and functional maturation. Bassoon in one of the first protein present at newly formed synaptic contacts. By electrophysiological approaches, we showed that Bassoon is important for the organization of the active zone during the first two postnatal weeks.Kainate receptors play an important role in the regulation of network activity during postnatal development. However, the impact of kainate receptors activation on synaptic maturation is less known. I showed a delay in functional maturation of mossy fiber synapses in mice deficient for the GluK2 subunit of kainate receptors (GluK2-/-). To know if this delay is correlated to morphological alterations of this synapse, we setup in vivo lentiviral infections of membrane fluorescent protein (YFP) in mouse pups (P1-P2). Using confocal microscopy and 3D reconstruction, we described the morphological maturation of mossy fiber synapses. We were able to correlate functional and morphological maturation and our results also showed an impairment in the formation of mossy fiber synapses in GluK2-/-. Together, these data reveal the importance of synaptic activity and of the coordination of pre- and post-synaptic assembly during synaptic maturation

Among the epileptic syndromes, temporal lobe epilepsy (TLE) is the most common form in adults. It is the consequence of a brain damage (viral infection, stroke, trauma, cancer ...), capable of triggering a cascade of events culminating in the appearance of spontaneous seizures that are, in many cases, difficult to control with the usual drug therapy. The period that elapses between the initial insult and the development of spontaneous recurrent seizures (SRSs) is defined "epileptogenesis”. The cellular and tissue changes that occur during this phase mainly interest the hippocampal region and include: neurodegeneration, neurogenesis, neuroinflammation and reactive gliosis, angiogenesis, and reorganization of brain circuits. Recently, it was shown that supplementation of neurotrophic factors (NTFs), such as FGF-2 (fibroblast growth factor-2) and BDNF (brain derived neurotrophic factor), has anti-epileptogenic effects by reducing neuronal death, favoring a correct neurogenesis and restoring a proper balance between excitatory and inhibitory circuits. In the main study reported in this thesis, we examined if this treatment can also affect neuroinflammatory processes. We used the pilocarpine model, in which an episode of status epilepticus (SE) is followed by an epileptogenic lesion. After three days, herpes viral vectors expressing FGF-2 and BDNF were injected in the hippocampus. Four, 11 and 25 days after treatment (DAI), animals were sacrificed and their brains removed to analyze the expression of three markers of inflammation: IL-1β, GFAP (a marker of astrocytosis), Ox42 (marker of microglia). The results show a very marked reduction of IL-1β expression, evident as early as 4 days after inoculation of the viral vector, and delayed, but significant, attenuation of the other two markers. The sprouting of mossy fibers is another characteristic of the epileptic hippocampal tissue, in which the axons of granule cells form excitatory synapses with cells not usually innervated, forming a circuit that may favour hyperexcitability. The results show that treatment with neurotrophic factors reduce aberrant sprouting of nerve fibers, in a way that correlates with the attenuation of cellular damage. Parallel behavioral studies have also highlighted the ability of the treatment to reduce the frequency and severity of SRSs that, in this model, begin to occur about 21 days after status epilepticus. Despite the promising results, clinical applicability of neurotrophic factors is limited by the choice of an appropriate route of administration. In the experiments reported in this thesis, herpes viral vectors have been used. These vectors were replication defective and engineered to express the two NTFs. However, their residual toxicity makes them unsuitable for human application. Stem cells modified to express genes of interest, including mesangioblasts (MABs), have demonstrated, in vitro, the ability to promote differentiation, survival and neuronal function. Last but not least, the ability to localize in the damaged site when systemically administered makes these cells viable alternatives to more invasive treatments. Although further investigations are required, the results collected in this thesis are an important contribution to the understanding of the multiple effects of NTFs. In addition, the characterization of an alternative and more applicable route of administration renders gene therapy with neurotrophic factor more applicable for the treatment of several neurodegenerative diseases.

Ho, Tsz Wan.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2009.
Includes bibliographical references (leaves 125-165).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.

Brain-derived neurotrophic factor (BDNF) is a modulator and mediator of structural and functional plasticity at synapses in the central nervous system. Despite our profound knowledge about the synaptic function of BDNF at synapses, it is still controversially discussed whether synaptic BDNF acts primarily from pre- or postsynaptic sites. In the central nervous system, several studies show that mossy fiber (MF) projections formed by hippocampal granule neurons store the highest amount of BDNF. However, immunofluorescence and RNA labelling studies suggest that MF BDNF is primarily produced by granule neurons. Multiple other studies prefer the view that BDNF is primarily produced by postsynaptic neurons such as CA3 pyramidal neurons. Here, we question whether the BDNF, which is stored in the mossy fiber synapse, is primarily produced by granule neurons or whether by other cells in the MF-CA3 microcircuit. After standardization of immunolabelling of BDNF, confocal imaging confirmed the localization of BDNF in presynaptic MF terminals. This anterograde location of synaptic BDNF was also found in distinct regions of the fear and anxiety circuit, namely in the oval nucleus of the bed nucleus stria terminals (ovBNST) and in the central amygdala. To find out whether the presynaptic BDNF location is due to protein translation in the corresponding presynaptic dentate gyrus (DG) granule neuron, we developed and characterized a mouse model that exhibits BDNF deletion specifically from adult DG granule neurons. In this mouse model, loss of presynaptic BDNF immunoreactivity correlated with the specific Creactivity in granule neurons, thus confirming that MF BDNF is principally released by granule neurons. After BDNF deletion from granule neurons, we observed more immature neurons with widely arborized dendritic trees. This indicated that local BDNF deletion also affects the local adult neurogenesis, albeit Cre-mediated BDNF deletion only occur in adult granule neurons. Since BDNF is a master regulator of structural synaptic plasticity, it was questioned whether it is possible to visualize presynaptic, synapse-specific, structural plasticity in mossy fiber synapses. It was established that a combination of Cre-techniques together with targeting of GFP to membranes with the help of palmitoylation / myristoylation anchors was able to distinctly outline the synaptic structure of the BDNF-containing MF synapse. In summary, the mouse model characterized in here is suited to investigate the synaptic signalling function of presynaptic BDNF at the mossy fiber terminal, a model synapse to investigate microcircuit information processing from molecule to behaviour
Der neurotrophe Wachstumsfaktor BDNF (brain-derived neurotrophic factor) ist ein Regulator und Vermittler von struktureller und funktionaler Plastizität in Synapsen des zentralen Nervensystems. Trotz des umfassenden Wissens über die synaptische Funktion von BDNF an Synapsen wird immer noch kontrovers diskutiert, ob synaptisches BDNF vorrangig von der prä- oder von der postsynaptischen Seite her agiert. Zahlreiche Studien zeigen, dass die größten BDNF Mengen des Zentralnervensystems in den Projektionen der hippocampalen Körnerzellen, den sogenannten Moosfasern (MF), enthalten sind. Während manche Studien basierend auf der Markierung von RNA und Immunofloureszenz nahelegen, dass MF BDNF in erster Linie von Körnerzellen produziert wird, bevorzugen zahlreiche andere Studien wiederum die Sicht, dass BDNF primär von postsynaptischen Neuronen wie beispielsweise den CA3 Pyramidenneuronen gebildet wird. In dieser Arbeit wurde die Fragestellung untersucht, ob das BDNF, welches in den Moosfasersynapsen enthalten ist, in erster Linie von Körnerzellen hergestellt wird, oder ob es hauptsächlich von anderen Zellen aus dem MF-CA3 Mikronetzwerk gebildet wird. Nachdem eine Standardisierung der Immunfluoreszenzmarkierung von BDNF etabliert wurde, konnte anhand von konfokaler Bildgebung die Lokalisierung von BDNF in den präsynaptischen MF Terminalen bestätiget werden. Diese anterograde Lokalisierung synaptischen BDNFs konnte außerdem in zwei weiteren Regionen des Furcht- und Angstnetzwerkes, genauer gesagt im ovalen Kern des bed nucleus stria terminalis (ovBNST) und in der zentralen Amygdala, nachgewiesen werden. Um Herauszufinden, ob die präsynaptische Lokalisation von BDNF von der Proteintranslation in den zugehörigen präsynaptischen Körnerzellen des Gyrus Dentatus abhängig ist, entwickelten und charakterisierten wir ein Mausmodel , welches die spezifische Deletion von BDNF aus den ausgereiften Körnerzellen des Gyrus Dentatus ermöglicht. In diesem Mausmodell korrelierte der Verlust präsynaptischer BDNF Immunreaktivität mit der spezifischen Cre-Aktivität in Körnerzellen, was bestätigt, dass MF BDNF hauptsächlich von den Körnerzellen ausgeschüttet wird. Nach BDNF Deletion aus den Körnerzellen konnten mehr unreife Neurone mit sich weit verzweigenden, dendritischen Strukturen beobachtet werden. Dies weist darauf hin, dass die lokale Deletion von BDNF auch die lokale adulte Neurogenese beeinflusst, obwohl die Crevermittelte BDNF Deletion nur in adulten Körnerzellen stattfindet. Da BDNF ein Hauptregulator von struktureller synaptischer Plastizität ist, kam die Frage auf, ob es möglich ist, diese präsynaptische, synapsenspezifische strukturelle Plastizität in Moosfasersynapsen zu visualisieren. Es wurde festgestellt, dass eine Kombination aus der Cre- Technik zusammen mit der gezielten Verankerung von GFP in der Zellmembran durch Palmitoylierungs-/Myristoylierungsmotive in der Lage ist, die synaptische Struktur von BDNF enthaltenden MF Synapsen darzustellen. Zusammenfassend konnte gezeigt werden, dass das hier entwickelte und charakterisierte Mausmodell dafür geeignet ist, die synaptische Signalfunktion präsynaptischen BDNFs in der Moosfaserterminale, einer Modellsynapse für die Erforschung der Informationsverarbeitung in Mikronetzwerken vom Molekül bis hin zum Verhalten, zu untersuchen

碩士
國立成功大學
藥理學研究所
89
Long-term potentiation (LTP) is characterized by its rapid induction and remarkable stability. In the past few years, most studies investigated the mechanism for its rapid induction. However, it is still unclear about the mechanisms underlying the conversion of initial potentiation into a persistent state. The consolidation of LTP plays a crucial role in memory formation, so it will help us to understand the mechanism of learning and memory. Therefore, the principle purpose of this study is to explore what factors and mechanisms contribute mechanism of the mossy fiber — CA3 LTP consolidation. The strategy of this study is to explore the causes of LTP reversal(depotentiation). Using mouse hippocampal slices, we have studied the induction of depotentiation of long-term potentiation (LTP) at the mossy fiber synapses onto CA3 pyramidal neurons. A long train of low-frequency (1 Hz/900 pulses) stimulation (LFS) induced a long-term depression of baseline synaptic transmission or depotentiation of previously established LTP, which was reversal and was independent of NMDA receptor activation. This LFS-induced depotentiation was observed when the stimulus was delivered 1 or 10 min after LTP induction. However, when LFS was applicated at 30 min after induction, significantly less depotentiation was found. The induction of depotentiation on one input was associated with a heterosynaptic reverse of the LTP previously induced on a separate pathway. In addition, this LFS-induced depotentiation appeared to be mediated by the activation of group 2 metabotropic glutamate receptors (mGluRs), because it was mimicked by bath-applied group 2 agonist (2S, 2’R, 3’R)-2-(2’, 3’-dicarboxycyclopropyl) glycine (DCG-IV) and was specifically inhibited by the group 2 antagonists, (S)-a-methyl-4-carboxyphenylglycine (MCPG) and (aS)-a-amino-a-(1S,2S)-2-carboxycyclopropyl-9H-xanthine-9-propanic acid (LY341495). Moreover, the induction of depotentiation was entirely normal when synaptic transmission was blocked by glutamate receptor antagonist kynurenic acid and was associated with a reversal of PPF attenuation during LTP expression. Pretreatment of the hippocampal slices with Gi/o protein inhibitor pertussis toxin (PTX) prevented the LFS-induced depotentiation. Additionally, the expression of mossy fiber LTP was not affected by removing of extracellular Ca2+ after LTP induction, suggesting that mossy fiber LTP dose not need persistent Ca2+ influx. Furthemore, we also found that dynorphin can mimic LFS to induce time dependent depotentiation. However, the opioid receptor antagonist naloxone, has no effect on LFS-induced depotentiation. These results suggest that LFS-induced depotentiation is primarly caused by glutamat-mediated cascade. Because protein phosphatase 2B inhibitor can also inhibit LFS-induced depotentiation, we predict that protein phosphatase 2B (PP2B) may also involve in LFS-induced depotentiation. In Westorn blotting experiments, we found that HFS-induced LTP is associated with an increase of rabphilin phosphorylation, and LFS significant reverses this effect. However, HFS-induced LTP did not cause significant any changes the phosphorylated state of Rim. Taken these results togrther, we proposed that activation of presynaptic group 2 mGluR and in turn triggering a PTX-sensitive Gi/o-protein-coupled signaling cascade and dephosphorylation of rabphilin may contribute to the LFS-induced depotentiation at the mossy fiber-CA3 synapses. However, the change of the phosphorylation state of Rim protein dose not involved on the LFS-induced LTP or LFS-induced depotentiation at mossy fiber-CA3 synapses.

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.

Tese de Doutoramento em Engenharia Biomédica, apresentada ao Departamento de Física da Universidade de Coimbra
The aim of this work was to study neuronal zinc changes, evoked by depolarizing media, at the hippocampal mossy fiber synapses from CA3 area. These synapses contain one of the largest concentrations of vesicular zinc in the central nervous system and also a high density of presynaptic ATP-sensitive potassium (KATP) channels. Zinc ions play a key role in numerous cellular processes including neurotransmission, the activation of metalloproteins and gene expression. At those synapses zinc is co-released with glutamate into the synaptic cleft, in a calcium dependent way, following electrically or chemically induced mossy fiber depolarization. Released zinc may then interact with various pre- and postsynaptic ionic and receptor-operated channels and have an important neuromodulatory role. In this study chemically evoked zinc signals were measured using the permeant form of the fluorescent zinc indicator Newport Green (Kd = 1 μM), at the zinc-enriched mossy fiber synapses from hippocampal slices (400 μm). In the first set of experiments (Part III), basal fluorescence signals from non-incubated and from Newport Green loaded slices, perfused with the normal extracellular medium, were detected. Their analysis reveals the existence of a constant autofluorescence component, representing approximately 75 % of the fluorescence intensity measured from incubated slices. A large fraction of this work addressed zinc changes induced by depolarizing KCl media. In order to determine the origin of these signals, antagonists of AMPA/KA and NMDA receptors and a blocker of voltage dependent calcium channels were applied extracellularly. The drugs used, NBQX (10 µM), D-APV (50 μM) and nifedipine (10 μM), respectively, largely decreased the amplitude of the KCl (60 mM) evoked signals, suggesting that they have a postsynaptic origin. In the presence of KCl (20 mM and 60 mM) solutions, the zinc transients exhibited a clear enhancement which, upon washout, was reduced or maintained, respectively. The signals associated with a second application of KCl (20 mM) were similar to those of the first one. However, the amount of steady potentiation observed for the KCl (60 mM) solution was maintained following washout, including during a second KCl (60 mM) application, but increased when it was combined with 1 mM ZnCl2. In the second group of experiments (Part IV), the effect of the voltage dependent potassium channel blocker tetraethylammonium (TEA), on zinc signals obtained with the same indicator, was studied. The TEA (25 mM) solution caused a depression of the zinc signals with respect to baseline, which recovered in the normal medium. It has been shown that released zinc activates the mossy fiber KATP channels, leading to rapid presynaptic membrane hyperpolarization. This should cause a reduction in glutamate and zinc release and, consequently, in postsynaptic zinc entry. This issue was tested comparing the zinc signals from two sets of consecutive TEA (25 mM) applications, one of which included the KATP channel inhibitor tolbutamide, The signals obtained in the presence of this drug had a smaller amplitude, supporting the idea that the KATP channels mediate, at least in part, the observed depression. In the last part of the work (Part V), flavoprotein associated autofluorescence changes, evoked by KCl (20 mM) and TEA (25 mM), were compared with those from NG incubated slices. The two depolarizing compounds, gave rise to opposite time varying autofluorescence signals, which were potentiated in the case of KCl and depressed for TEA, as found for the zinc signals, suggesting the existence of a close relationship between the autofluorescence and zinc changes. The measurements from NG loaded slices include a time variable autofluorescence component and a NG-zinc component. Under the experimental conditions used the first component is considered to have flavoprotein origin, reflecting FAD levels and the redox metabolic state of mitochondria. The real zinc signal was obtained subtracting, point to point, the autofluorescence from the total fluorescence signals. In the presence of tolbutamide the calculated zinc changes are largely reduced with respect to those obtained without the blocker. This fact supports the idea that, at the hippocampal mossy fiber synapses, a KATP mediated zinc action contributes to the neuroprotective role of zinc under conditions of intense synaptic stimulation.
O objetivo deste trabalho consistiu no estudo de variações neuronais de zinco, induzidas por meios despolarizantes, nas sinapses das fibras musgosas da área CA3 do hipocampo. Estas sinapses contêm uma das maiores concentrações de zinco vesicular no sistema nervoso central e também uma elevada densidade de canais pré-sinápticos de potássio sensíveis ao ATP (KATP). Os iões de zinco têm um papel fundamental em inúmeros processos celulares, incluindo a neurotransmissão, a activação de metaloproteínas e a expressão genética. Naquelas sinapses o zinco é co-libertado com o glutamato na fenda sináptica, duma forma dependente do cálcio, após a despolarização das fibras musgosas induzida elétrica- ou quimicamente. O zinco libertado pode então interagir com diversos canais iónicos e de receptores, pré- e pós-sinápticos e ter um papel neuromodulador importante. Neste estudo mediram-se sinais de zinco induzidos quimicamente usando a forma permeante do indicador de zinco fluorescente Newport Green (Kd = 1 µM), nas sinapses das fibras musgosas de fatias do hipocampo (400 µm), que são ricas em zinco. No primeiro conjunto de experiências (Parte III) foram detetados sinais de fluorescência basal, de fatias não incubadas ou contendo o indicador Newport Green, perfundidas com o meio extracelular normal. A sua análise revela a existência de uma componente constante de autofluorescência, representando aproximadamente 75% da intensidade de fluorescência das fatias incubadas. Uma grande parte deste trabalho abordou variações de zinco induzidas por meios de KCl despolarizantes. A fim de determinar a origem destes sinais, aplicaram-se extracelularmente antagonistas de receptores de AMPA/KA e NMDA e um bloqueador de canais de cálcio dependentes do potencial. Os compostos utilizados, respetivamente NBQX (10 µM), D-APV (50 µM) e nifedipine (10 µM), reduziram bastante a amplitude dos sinais induzidos por KCl (60 mM), sugerindo que eles têm uma origem pós-sináptica. Na presença de soluções de KCl (20 mM e 60 mM) os sinais de zinco exibiram um aumento claro que, após a remoção da solução foi, respetivamente reduzido ou mantido. Os sinais associados a uma segunda aplicação de KCl eram semelhantes aos da primeira. No entanto, o valor da potenciação estacionária observada para a solução de KCl (60 mM) manteve-se após a sua remoção, incluindo durante uma segunda aplicação de KCl (60 mM), mas aumentou quando ela incluía 1 mM ZnCl2. No segundo grupo de experiências (Parte IV), estudou-se o efeito do bloqueador dos canais de potássio dependents do potencial tetraetilamónio (TEA), em sinais dezinco obtidos como mesmo indicador. A solução de TEA (25 mM) originou uma depressão dos sinais de zinco relativamente à linha base, que recuperou no meio normal. Mostrou-se já que o zinco libertado ativa os canais de KATP das fibras musgosas, levando rapidamente a uma hiperpolarização da membrana pre-sinática. Isto deveria causar uma redução na libertação de glutamate e de zinco e, consequentemente, na entrada de zinco pós-sinático. Esta questão foi testada comparando os sinais de zinco de dois conjuntos de aplicações consecutives de TEA (25 mM), uma ds quais incluía tolbutamida que é um inibidor dos canais de KATP. Os sinais obtidos na presença deste composto tinham menor amplitude, de acordo com a ideia que a depressão observada é, pelo menos em parte, mediada pelos canais de KATP. Na última parte do trabalho (Parte V), compararam-se variações de autofluorescência associadas com flavoproteínas, induzidas por KCl (20 mM) e TEA (25 mM), com as obtidas de fatias incubadas com NG. Os dois compostos despolarizantes, originaram sinais opostos de autofluorescênica variáveis no tempo, potenciados no caso de KCl e inibidos por TEA, tal como se verificou para os sinais de zinco, sugerindo a existência de de uma relação próxima entre as variações de autofluorescência e de zinco. As medições efetuadas em fatias contendo NG incluem uma componente de autofluorescência variável no tempo e uma componente de zinco-NG. Nas condições experimentais usadas considera-se que a primeira componente tem origem em flavoproteínas, refletindo níveis de FAD e o estado metabólico redox das mitocôndrias. O sinal real de zinco foi obtido subtraindo, ponto por ponto, a autofluorescência dos sinais de fluorescência total. Na presença de tolbutamida as variações de zinco calculadas são muito reduzidas em relação às obtidas sem o bloqueador. Este facto está de acordo com a ideia que, nas sinapses das fibras musgosas do hipocampo, a ação do zinco mediada pelos canais de KATP, contribui para o papel neuroprotetor do zinco em condições de estimulação sináptica intensa.