Made available in DSpace on 2014-12-17T15:28:52Z (GMT). No. of bitstreams: 1 HermanyMV_DISSERT.pdf: 1765987 bytes, checksum: dedd84315ca1c69652d27407ffd67d85 (MD5) Previous issue date: 2013-03-04 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Recently, genetically encoded optical indicators have emerged as noninvasive tools of high spatial and temporal resolution utilized to monitor the activity of individual neurons and specific neuronal populations. The increasing number of new optogenetic indicators, together with the absence of comparisons under identical conditions, has generated difficulty in choosing the most appropriate protein, depending on the experimental design. Therefore, the purpose of our study was to compare three recently developed reporter proteins: the calcium indicators GCaMP3 and R-GECO1, and the voltage indicator VSFP butterfly1.2. These probes were expressed in hippocampal neurons in culture, which were subjected to patchclamp recordings and optical imaging. The three groups (each one expressing a protein) exhibited similar values of membrane potential (in mV, GCaMP3: -56 ?8.0, R-GECO1: -57 ?2.5; VSFP: -60 ?3.9, p = 0.86); however, the group of neurons expressing VSFP showed a lower average of input resistance than the other groups (in Mohms, GCaMP3: 161 ?18.3; GECO1-R: 128 ?15.3; VSFP: 94 ?14.0, p = 0.02). Each neuron was submitted to current injections at different frequencies (10 Hz, 5 Hz, 3 Hz, 1.5 Hz, and 0.7 Hz) and their fluorescence responses were recorded in time. In our study, only 26.7% (4/15) of the neurons expressing VSFP showed detectable fluorescence signal in response to action potentials (APs). The average signal-to-noise ratio (SNR) obtained in response to five spikes (at 10 Hz) was small (1.3 ? 0.21), however the rapid kinetics of the VSFP allowed discrimination of APs as individual peaks, with detection of 53% of the evoked APs. Frequencies below 5 Hz and subthreshold signals were undetectable due to high noise. On the other hand, calcium indicators showed the greatest change in fluorescence following the same protocol (five APs at 10 Hz). Among the GCaMP3 expressing neurons, 80% (8/10) exhibited signal, with an average SNR value of 21 ?6.69 (soma), while for the R-GECO1 neurons, 50% (2/4) of the neurons had signal, with a mean SNR value of 52 ?19.7 (soma). For protocols at 10 Hz, 54% of the evoked APs were detected with GCaMP3 and 85% with R-GECO1. APs were detectable in all the analyzed frequencies and fluorescence signals were detected from subthreshold depolarizations as well. Because GCaMP3 is the most likely to yield fluorescence signal and with high SNR, some experiments were performed only with this probe. We demonstrate that GCaMP3 is effective in detecting synaptic inputs (involving Ca2+ influx), with high spatial and temporal resolution. Differences were also observed between the SNR values resulting from evoked APs, compared to spontaneous APs. In recordings of groups of cells, GCaMP3 showed clear discrimination between activated and silent cells, and reveals itself as a potential tool in studies of neuronal synchronization. Thus, our results indicate that the presently available calcium indicators allow detailed studies on neuronal communication, ranging from individual dendritic spines to the investigation of events of synchrony in neuronal networks genetically defined. In contrast, studies employing VSFPs represent a promising technology for monitoring neural activity and, although still to be improved, they may become more appropriate than calcium indicators, since neurons work on a time scale faster than events of calcium may foresee / Neur?nios se comunicam por meio de sinapses, trocando mensagens capazes de modificar o potencial de membrana de outros neur?nios. Demonstrar o papel desses sinais e decodificar essa linguagem el?trica representa o grande objetivo da neuroci?ncia moderna. Atualmente, a eletrofisiologia ? o ramo da neuroci?ncia capaz de investigar esses recursos el?tricos de neur?nios - que v?o desde registros de condut?ncia e comportamento cin?tico de canais i?nicos individuais at? a demonstra??o de neur?nios individuais implicados em comportamentos complexos. Nesse sentido, diferentes estados cerebrais e comportamentos implicam o recrutamento de grandes conjuntos de neur?nios se comunicando em um estado coerente, din?mico. Al?m disso, essas grandes popula??es s?o formadas por diversos subtipos neuronais cuja an?lise requer t?nicas que possibilitem uma resolu??o temporal e espacial de c?lulas individuais e, prefencialmente, de subtipos espec?ficos. Apenas recentemente, indicadores ?pticos geneticamente codificados surgiram como ferramentas n?o invasivas de alta resolu??o espacial e temporal utilizados para monitorar a atividade de neur?nios individuais e popula??es neuronais espec?ficas. O n?mero crescente de novos indicadores optogen?ticos, juntamente com a aus?ncia de compara??es em condi??es id?nticas, gerou dificuldade em escolher a mais adequada das prote?nas, dependendo do desenho experimental. Portanto, o objetivo deste estudo foi comparar tr?s prote?nas rep?rter recentemente desenvolvidas: os indicadores de c?lcio GCaMP3 e R-GECO1, e o indicador de voltagem VSFP butterfly1.2. Foram expressos em neur?nios do hipocampo em cultura, os quais foram submetidos a registros de patch-clamp e de imageamento ?ptico. Os tr?s grupos (cada um expressando uma prote?na) exibiram valores semelhantes de potencial de membrana (em mV, GCaMP3: -56 ? 8,0; R-GECO1: -57 ? 2,5; VSFP: -60 ? 3,9; p = 0,86), no entanto, o grupo de neur?nios que expressam VSFP mostrou uma m?dia mais baixa de resist?ncia de entrada do que os outros grupos (em Mohms, GCaMP3: 161 ? 18,3; GECO1-R: 128 ? 15,3; VSFP: 94 ? 14,0; p = 0,02). Cada neur?nio foi submetido a inje??es de correntes com frequ?ncias diferentes (10 Hz, 5 Hz, 3 Hz, 1,5 Hz, e 0,7 Hz) e as suas respostas de fluoresc?ncia foram registradas. Em nosso estudo, apenas 26,7% (4/15) dos neur?nios que expressam VSFP mostraram sinal de fluoresc?ncia detect?vel em resposta a potenciais de a??o. O valor m?dio de sinal-para-ru?do (SNR), obtido em resposta a cinco potenciais de aҫ?o (a 10 Hz) foi pequeno (1,3 ? 0,21), no entanto a cin?tica r?pida do VSFP permite a discrimina??o de disparos, como picos individuais, com detec??o de 53% dos APs evocados. Freq??ncias abaixo de 5 Hz, assim como variaҫ?es no potencial de membrana subliminares, foram indetect?veis devido ao alto ru?do do sinal de fluoresc?ncia. Por outro lado, os indicadores de c?lcio mostraram maior altera??o na fluoresc?ncia, seguindo o mesmo protocolo (cinco potenciais de aҫ?o a 10 Hz). Entre os neur?nios expressando GCaMP3, 80% (8/10) exibiram sinal, com um valor m?dio de SNR de 21 ? 6,69 (soma), enquanto que para os neur?nios expressando R-GECO1, 50% (2/4) dos neur?nios demonstraram sinal com um valor m?dio SNR de 52 ? 19,7 (soma). Para protocolos de 10 Hz, 54% dos disparos foram detectados com evocado GCaMP3 e 85% com o R-GECO1. Disparos foram detectados em todas as frequ?ncias e os sinais de fluoresc?ncia foram tamb?m detectados a partir de despolariza??es subliminares. Sendo GCaMP3 o indicador mais prov?vel de produzir sinal de fluoresc?ncia e com alto SNR, alguns experimentos foram realizados somente com essa prote?na. Observamos que GCaMP3 ? eficaz na detec??o de inputs sin?pticas (envolvendo influxo de Ca2+), com alta resolu??o espacial e temporal. Tamb?m foram observadas diferen?as entre os valores de SNR resultantes dos disparos evocados, em compara??o com os disparos espont?neos. Em registros de grupos de c?lulas, GCaMP3 mostrou clara discrimina??o entre c?lulas ativadas e sil?ncio, revelando-se como uma ferramenta potencial em estudos de sincroniza??o neuronal. Assim, nossos resultados sugerem que os indicadores de c?lcio dispon?veis atualmente permitem estudos detalhados sobre a comunica??o neuronal, que v?o desde dendritos individuais at? a investiga??o de eventos de sincronia em redes neuronais geneticamente definidas. Em contraste, VSFPs representam uma tecnologia promissora para monitorar a atividade neural e, apesar de ainda requererem melhoramentos, podem se tornar mais apropriados do que os indicadores de c?lcio, uma vez que os neur?nios trabalham em uma escala de tempo mais r?pida do que eventos de c?lcio podem prever

En dépit de l'abondance de données scientifiques, les mécanismes cellulaires régulant la sécrétion du pic pré-ovulatoire de LH lors du proestrus, restent encore mal compris.Afin de pouvoir étudier les mécanismes sous-jacents à cette sécrétion, j’ai tout d’abord adapté des techniques innovantes d’imagerie fonctionnelle en microscopie de fluorescence in vivo, d’injections de vecteurs viraux dans l’hypophyse, d’optogénétique sur animal éveillé et d’immunohistofluorescence sur organe entier.J’ai ensuite montré la plasticité structurelle des cellules gonadotropes et des péricytes (cellules « murales » péri-vasculaires) lors du proestrus sur des hypophyses transparisées. Ce remodelage a permis de proposer l’existence d’une unité Gonadotrope-Vasculaire (GVU) composée des cellules gonadotropes, des capillaires fenêtrés et des péricytes dans laquelle ces derniers moduleraient le pic pré-ovulatoire de LH.La contraction des péricytes via l’activation de la Channelrhodopsine-2 a permis de mettre en évidence leur rôle dans la potentialisation de la sécrétion de LH chez des animaux libres de leurs mouvements et implantés d’une fibre optique.Des expériences de microscopie à l’aide d’une lentille GRIN implantée au-dessus de l’hypophyse ont permis, chez l’animal éveillé en configuration « tête-fixée », d’étudier le flux sanguin et l’activité calcique de cellules de la GVU exprimant GCaMP6. Cette étude a également été menée sur la face ventrale de l’hypophyse sur souris anesthésiée. Les résultats montrent une activité calcique in vivo augmentée dans les cellules endocrines hypophysaires et diminuée dans les péricytes lors d’une sécrétion de la LH induite par la GnRH. / In spite of abundance of scientific data, cellular mechanisms regulating the secretion of the pre-ovulatory LH surge during proestrus are still poorly understood.In order to study the mechanisms underlying this secretion, I adapted innovative tech-niques for in vivo fluorescence functional imaging, injection of viral vectors in the pitui-tary gland, optogenetics in awake animals and immunohistofluorescence in the whole organ.I then showed structural plasticity of gonadotroph cells and pericytes (perivascular "mural" cells) during proestrus in cleared hypophyses. This suggested the existence of a Gonadotroph-Vascular Unit (GVU) composed of gonadotroph cells, fenestrated capil-laries and pericytes, in which the latter would modulate the pre-ovulatory LH surge.Pericytes contraction via Channelrhodopsine-2 activation permitted to demonstrate their role in the sensitization of LH secretion in freely moving animals implanted with an optical fiber.Finally, blood flow and calcium activity in GVU cells expressing GCaMP6 were performed in awake « head-fixed » animals in which visualization of the pituitary gland was achievable through an implanted GRIN lens. These experiments were also conduct-ed at the ventral side of the pituitary gland in anesthetized mice. Analysis showed that in vivo calcium activity increases in endocrine cells and decreases in pericytes during GnRH-induced LH secretion.

L’exposition aux radiations laser infrarouge peut être utilisée afin de dépolariser des neurones et stimuler l’activité neuronale. Le mécanisme sous-jacent d’une telle stimulation est supposé résulter d’une interaction photothermique. En effet, l’absorption de la radiation infrarouge par le tissu biologique cible, et l’eau qu’il contient, induit une augmentation de température de manière localisée, qui soit influencerait directement les propriétés membranaires de la cellule soit agirait par le biais de l’activation de canaux ioniques thermo-sensibles. Dans la plupart des cas, l’activité électrique des neurones est mesurée électriquement à l’aide de microélectrodes, mais elle peut également être sondée par le biais de la microscopie de fluorescence faisant intervenir des indicateurs calciques. Dans ce travail, l’impact de l’exposition à la radiation infrarouge sur les signaux calciques de neurones a été étudié dans le but d’éclaircir et de préciser le mécanisme résultant de l’interaction photothermique. Des neurones HT22, issus d’hippocampe de souris, et des cellules U87, issues d’un glioblastome humain, ont été utilisés en tant qu’exemples de cellules électriquement excitables et non excitables respectivement. Afin de mesurer la température et les signaux calciques au niveau cellulaire, les fluorophores Rhodamine B et Fluo-4 ont été employés. Le montage, par conséquent tout optique, pour étudier l’influence de l’exposition infrarouge sur l’activité neurale est donc présenté, ainsi que la démarche scientifique menant à l’identification de l’implication de l’activité de la phospholipase C dans le mécanisme étudié. La possibilité de stimuler l’activité neurale in vivo, dans le cerveau d’une souris, avec une mesure simultanée des signaux calciques, est également démontrée à l’aide de souris transgéniques exprimant le GCaMP6S. / Infrared laser light radiation may be used to depolarize neurons and to stimulate neural activity. The underlying mechanism of such stimulation is believed to happen due to a photothermal interaction. The absorption of the infrared radiation by the targeted biological tissue inducing a local temperature increase which either directly influence membrane properties or act via temperature sensitive ion channels. Action potentials are typically measured electrically in neurons with microelectrodes, but they can also be observed using fluorescence microscopy techniques that use synthetic or genetically encoded calcium indicators. In this work, we studied the impact of infrared laser light on neuronal calcium signals to address the mechanism of these thermal effects. HT22 mouse hippocampal neurons and U87 human glioblastoma cells were used loaded with the fluorescent calcium dye Fluo-4 and with the temperature sensitive fluorophore Rhodamine B to measure calcium signals and temperature changes at the cellular level. Here we present our all-optical strategy for studying the influence of infrared laser light on neural activity, and the scientific approach leading to conclusion of the involvement of Phospholipase C activity during infrared neural stimulation. The ability of infrared exposure to trigger neural activity in mice brain in vivo is also investigated with the use of GCaMP6s transgenic mice.

The majority of fast excitatory neurotransmission in the brain occurs at glutamatergic synapses. The extensive dendritic arborisations of pyramidal neurons in the neocortex and hippocampus harbor thousands of synaptic connections, each formed on tiny protrusions called dendritic spines. Spine synapses are rapidly established during early postnatal development – a key period in neural circuit assembly – and are subject to dynamic activity-dependent plasticity mechanisms that are believed to underlie neural information storage and processing for learning and memory. Recent decades have seen remarkable progress in identifying diverse plasticity mechanisms responsible for regulating synapse structure and function, and in understanding the processes underlying computation of synaptic inputs in the dendrites of individual neurons. These advances have strengthened our understanding of the biological mechanisms underlying brain function but, not surprisingly, they have also raised many new questions. Using a combination of whole-cell electrophysiology, 2-photon imaging and glutamate uncaging in rodent brain slice preparations, I have helped to document the subtype-specific regulation of glutamate receptors during a homeostatic form of synaptic plasticity at CA1 pyramidal neurons of the hippocampus, and have discovered novel synaptic calcium dynamics during a critical period of neural circuit formation. First, we found that during a homeostatic response to prolonged inactivity, both AMPA and NMDA subtypes of glutamate receptors undergo a switch in subunit composition at synapses, but exhibit a divergence in their subcellular localization at extrasynaptic regions of the plasma membrane (this work was published in the Journal of Neuroscience in 2013). In separate series of experiments using 2-photon calcium imaging, I discovered a functional coupling between NMDA receptor activation and intracellular calcium release at dendritic spines and dendrites that is selectively expressed during a critical period of synapse formation. This synaptic calcium signaling mechanism enabled the transformation of distinct spatiotemporal patterns of synaptic input into salient biochemical signals, and is thus apt to locally regulate synapse development along individual dendritic branches. Consistent with this hypothesis, I found evidence for non-random clustering of synapse development between neighboring dendritic spines. Together, these experimental results expand the current understanding of the dynamics of synapse function during homeostatic plasticity and early postnatal development. --- Les synapses glutamatergiques soutiennent la majorité de la neurotransmission excitatrice rapide du cerveau. Des milliers de ces synapses, localisées sur de minuscules saillies appelées épines dendritiques, décorent les vastes arborisations dendritiques des neurones pyramidaux du néocortex et de l'hippocampe. Ces synapses sont formées tôt lors du développement postnatal et sont soumises à des mécanismes dynamiques de plasticité qui sous-tendent, croit-on, les capacités d'apprentissage et de mémoire du cerveau. Les dernières décennies ont vu des progrès remarquables dans l'identification de divers mécanismes de régulation de la structure et de la fonction des synapses sur différentes échelles de temps, et dans la compréhension des processus qui régissent l’intégration des inputs synaptiques au niveau des dendrites individuelles. Ces progrès ont renforcé notre compréhension des éléments fondamentaux régissant la fonction cérébrale et ont ouvert de nouvelles voies d’investigations neurophysiologiques. En utilisant une combinaison d’électrophysiologie cellulaire, d'imagerie à deux-photons et de photolibération de glutamate sur des neurones pyramidaux de la région CA1 de l'hippocampe de rats, j’ai contribué à la découverte et à la caractérisation de nouvelles régulations des récepteurs du glutamate durant la plasticité synaptique homéostatique. J’ai également découvert un nouveau type de dynamique de calcium synaptique relié à une organisation spatiale du développement des synapses pendant une période critique de l’ontogénie des circuits neuronaux. Dans la première étude, nous avons constaté que lors d'une plasticité de type homéostatique induite par une inactivité prolongée, les récepteurs de glutamate de types AMPA et NMDA sont soumis à un changement important dans la composition de leurs sous-unités. De plus, nous avons observé un ciblage différentiel de ces récepteurs vers des compartiments subcellulaires spécifiques des neurones. Dans une série d'expériences séparée utilisant l’imagerie calcique à deux-photons, j’ai découvert un couplage fonctionnel durant le développent entre l'activation des récepteurs NMDA et une libération de calcium intracellulaire qui envahit tant les épines dendritiques que les dendrites. J’ai également trouvé que ce mécanisme de signalisation de calcium synaptique transforme des motifs spatiotemporels d’activités synaptiques spécifiques en signaux biochimiques post-synaptiques de manière à potentiellement réguler l’organisation spatiale des synapses durant le développement. Conformément à cette hypothèse, j’ai observé des manifestations fonctionnelles claires de regroupement dans l’espace de synapses de forces similaires le long de branches dendritiques individuelles. Ensemble, ces résultats expérimentaux élargissent notre compréhension actuelle de de la fonction des synapses durant la plasticité homéostatique ainsi que durant le développement postnatal du cerveau. En étudiant les mécanismes neurophysiologiques de base, il sera possible d'avoir un aperçu plus profond du fonctionnement du cerveau et de ses pathologies.

Les techniques optogénétiques ont laissé entrevoir un potentiel exceptionnel dans l’étude des mécanismes gouvernant l’intégration de l’information dans le système nerveux. Afin d’établir les relations existant entre des séquences d’activité neuronale définies et le comportement, les techniques optiques doivent permettre d’appréhender des groupes de neurones avec une résolution cellulaire chez l’animal éveillé et mobile. Jusqu’alors, l’activité neuronale chez l’animal vigile non contraint n’a été contrôlée que via l’illumination en champs large. Dans ce travail, nous démontrons une résolution proche de la cellule unique pour la photoactivation, chez l’animal éveillé et mobile, à l’aide d’un fibroscope. Les motifs de photoactivation, produits par holographie, sont transmis jusqu’à la souris par un guide d’image couplé à un micro-objectif. Une imagerie de fluorescence permet de localiser les cellules d’intérêt et d’enregistrer l’activité neuronale, par épifluorescence, illumination structurée, ou encore microscopie confocale multi-point sans balayage. Le fibroscope est testé chez l’animal anesthésié et chez l’animal éveillé et mobile, dont les interneurones de la couche moléculaire du cervelet expriment les protéines ChR2-tdTomato et GCaMP. Nous avons généré des signaux calciques somatiques en ciblant les corps cellulaires avec des spots de photoactivation de 5µm de diamètre. Chez l’animal anesthésié, nous avons démontré que la photoactivation pouvait être réalisée avec une résolution latérale de 10µm et une résolution axiale de 40µm, en considérant la demi-largeur à mi-hauteur de la courbe de résolution. Nous avons montré qu’un ou plusieurs soma pouvaient être ciblés sélectivement. Chez l’animal éveillé et mobile, le champs de vue est resté stable au cours des acquisitions. Nous avons trouvé une résolution latérale pour la photoactivation égale à 10µm, démontrant une résolution de photoactivation proche de la cellule unique chez l’animal vigile non contraint. / Optogenetics has shown great potential to study the mechanisms governing information integration in the brain. To link specific spatiotemporal activity patterns and behaviours, optical methods should provide simultaneous access to a group of neurons with single cell resolution in freely behaving animals. So far, however, optogenetic control of neural activity in freely behaving rodents has been performed with widefield illumination only. Here, we demonstrate photoactivation with near-cellular resolution in freely behaving mice using a fiberscope. Photoactivation patterns, produced with computer-generated holography, were transmitted to the mouse using a fiber bundle coupled to a micro-objective. Fluorescence imaging allowed locating cells and recording neuronal activity, via either epifluorescence, structured illumination, or scanless multi-point confocal microscopy. The fiberscope was tested both in anesthetized and freely-behaving mice co-expressing ChR2-tdTomato and GCaMP proteins in cerebellar molecular layer interneurons. By targeting an interneuron soma with a 5µm diameter photoactivation spot, we could elicit a calcium transient. In anesthetized animals, we demonstrated that photoactivation could be performed with 10µm and 40µm lateral and axial resolution, half-width at half maximum, respectively. We showed that either a single or multiple somata could be selectively targeted. In awake unrestrained animals, the field of view remained stable during our acquisitions. We found that photoactivation lateral resolution remained equal to 10µm, demonstrating photoactivation with near-cellular resolution in freely behaving mice.

L’ischémie cérébrale est caractérisée par une interruption totale ou partielle de l’apport sanguine au cerveau, conduisant à une privation d’oxygène et de glucose pour les cellules du cerveau. La série de processus cellulaires qui sont déclenchées par une ischémie cérébrale sont nombreux et complexes. La réduction sévère d’oxygène et de glucose la diminution de la production d’ATP et un changement drastique de la concentration de K+, du pH intracellulaire et extracellulaire et de la production de lactate. La perturbation du métabolisme énergétique au sein des tissus ischémiés conduit rapidement à la dépolarisation membranaire et au relarguage de neurotransmetteurs dans le milieu extracellulaire. Dans le cervelet, l’impact d’un stress ischémique à largement été étudié sur les cellules de Purkinje, seule voie de sortie neuronale du cortex cérébelleux. Il a été montré que le glutamate, relargué par une surexcitation des fibres glutamatergique et par l’inversion des transporteurs du glutamate, est la cause principale de la dépolarisation anoxique des cellules de Purkinje. Cependant, la compréhension de la réponse astrocytaire et l’influence des astrocytes vis-à-vis de l’ischémie ne sont pas encore connu.La cellule de Bergmann est un astrocyte radiaire qui compose un réseau couplé électriquement, formant des interactions anatomiques et fonctionnelles complexes avec les neurones du cortex cérébelleux. En utilisant un modèle in vitro d’ischémie cérébrale, la privation d’oxygène et de glucose (OGD), plusieurs caractéristiques de base de la réaction astrocytaire à l'ischémie sont analysés. Des expériences en patch clamp et d’imagerie calcique sont réalisées sur tranche de cervelet adulte révélant la réponse de la glie de Bergmann à l’OGD par une dépolarisation progressive de la membrane, avec en parallèle une augmentation de calcium cytosolique soutenue. L’enregistrement apparié entre cellule de Purkinje et cellule de Bergmann révèle des différences importantes de réponse à l’OGD entre ces deux types cellulaires. De plus, nous avons mesuré les changements de la concentration de K+ extracellulaire durant l’OGD en utilisant des microélectrodes sensibles aux ions. Nos résultats montrent une corrélation importante entre la dynamique du K+ extracellulaire et la dépolarisation membranaire de la cellule de Bergmann au cours de l’OGD. / Cerebral ischemia is characterized by partial or total interruption of the blood supply to the brain resulting in glucose and oxygen deprivation to brain cells. The series of cellular processes that are unleashed by cerebral ischemia are complex. The severe reduction in oxygen and glucose induces decreases in ATP production and dramatic changes in extracellular K concentration, pH of intracellular and extracellular space and lactate production. The disruption of energy metabolism in the ischemic tissue rapidly lead to membrane depolarisation and neurotransmitters are released into the extracellular space. In the cerebellum, the impact of an ischemic stress has been extensively studied in Purkinje cells, the only neuronal output of the cerebellar cortex. It has been shown that glutamate released from overexcited fibers and from reversal of glutamate transporters, is the principal cause of the dramatic, anoxic depolarization in Purkinje cells. However a detailed understanding of the astrocytic response to cerebellar ischemia and the potential influence of astrocyte to ischemia outcome is still lacking.Bergmann glia (BG) are radial gial cells that form networks of electrically coupled cells underling complex anatomical and functional interactions with the neurons of the cerebellar cortex. Using an in vitro model of cerebral ischemia, the oxygen and glucose deprivation (OGD), several basic features of astrocytic reaction to ischemia are analyzed. Patch clamp and calcium imaging experiments performed in cerebellar slices from adult mice revealed that BG respond to OGD with a progressive membrane depolarisation that is paralleled with a sustained cytosolic calcium increase. Double patch-clamp recordings between Purkinje neurons and BG reveal different responses to OGD in these cell types. Furthermore, we measured extracellular potassium concentration changes during OGD by using ion-sensitive microelectrodes. Our results indicate an important correlation between the BG membrane depolarisation and the extracellular K dynamics during OGD.

Modulation of a synaptic transmission in the spinal cord dorsal horn plays a key role in nociceptive signalling, especially in states of pathological pain. The goal of this study was to develop a method for calcium imaging in spinal cord slices in vitro. This method allowed us to record changes of intracellular free calcium ions concentration (iCa2+ ), that are a major mediator of neuronal plasticity. In this work, we have focused on application of this method in a conventional fluorescence microscope and on the role of different neuromodulators of synaptic activity. Changes of iCa2+ induced by dorsal root electrical stimulation were recorded altogether in 744 dorsal horn (lamina I and II) neurons. In the first series of experiments, stimulation protocols activating preferentially A and A + C dorsal root fibers were used and long-term stability of the calcium responses was verified. The dorsal root stimulation induced in the neurons fast and delayed type of calcium response. Application of AMPA and NMDA receptors antagonists, CNQX (50μM) and MK801 (45μM), reduced the calcium response amplitude and confirmed the importance of glutamate receptors in synaptic activation. In several experiments the effect of capsaicin a TRPV1 receptors agonist, application was tested. Application of even low...

<p>Dendritic spines are mushroom-shaped appendages on the dendritic branches of neurons. They are invaluable to the function of the brain as they form the major site for excitatory signal transmission in the mammalian brain. These ubiquitous structures have several invaluable and unique characteristics – namely that their morphological and functional characteristics are activity-dependent and undergo remodeling as the spine experiences stimulation. This activity-dependent regulation then in turn modulates the excitatory postsynaptic potential that propagates into the adjacent parent dendrite, and which ultimately reaches the somatic compartment. The mediation of this modulatory effect on the postsynaptic signal by dendritic spines renders them invaluable to the brain’s ability to change neuronal circuits as it learns. The relationship between the structural and functional change in dendritic spines as plasticity is induced remains poorly understood; while efforts have been made to examine the morphology of dendritic spines during plasticity as well as the change to receptor insertion on the postsynaptic density, a comprehensive methodology to interrogate the concomitant changes to several aspects of dendritic spine structure and function as plasticity occurs has not been established. In this study, such a methodology was developed in order to facilitate future study of how a dendritic spine’s diffusional neck resistance, head volume, calcium-sensitive channels (on the postsynaptic density), and excitatory postsynaptic potential amplitude change concurrently as the spine undergoes activity-dependent regulation. This activity-dependent regulation also occurs in groups of spines called “clusters” <em>in vivo</em>, and the structural and functional dynamics of spines as these groups are formed also remains unknown. In order to to facilitate future <em>in vivo</em> studies on how clustered dendritic spines may change dynamically in both structure and function, a methodology for surgically accessing and recording calcium-based activity from the primary auditory cortex was developed, as the frequency-specific tuning of dendritic spines in this cortical area forms a compelling environment in which to study the relationship between spine form and function. </p>

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