I present applications of imaging and spectroscopy to understand mechanical, chemical, and electrical dynamics in biological materials. The first part describes the development and characterization of a protein-based fluorescent calcium and voltage indicator (CaViar). The far-red fluorescence of CaViar faithfully tracks the cardiac action potential in cardiomyocytes. CaViar's green fluorescence reports the resulting calcium transients. I demonstrated the applicability of CaViar in vivo with transgenic zebrafish designed to express CaViar in their hearts. Spinning disk confocal imaging allowed detailed three-dimensional mapping of simultaneous voltage and calcium dynamics throughout the heart of zebrafish embryos, in vivo, as a function of developmental stage. I tested the effect of channel blockers on voltage and calcium dynamics and discovered a chamber-specific transition from a calcium-dependent to a sodium-dependent action potential. I also describe a new measurement technique using a fluorescent voltage indicator to report absolute voltage via the indicator's temporal response. / Physics

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The hippocampal CA1 is crucial for myriad types of learning and memory. It is theorized to provide a spatiotemporal framework for the encoding of relevant information during learning, allowing an individual to create a cognitive map of its environment and experiences. To probe CA1 network dynamics that underlie such complex cognitive function, in this work we used recently developed cellular optical imaging techniques that provide high spatial and temporal resolutions. Genetically-encoded calcium indicators offer the ability to record intracellular calcium dynamics, a proxy of neural activity, from hundreds of cells in behaving animals with single cell resolution in genetically-defined cell types. In complement, recently developed genetically-encoded voltage indicators have enabled direct recording of transmembrane voltage of individual genetically-defined cells in behaving animals. The work presented here uses the genetically-encoded calcium indicator GCaMP6f and the genetically-encoded voltage indicator SomArchon to interrogate the activities of individual hippocampal CA1 neurons and their relationship to the dynamics of the broader network during behavior. First, we provide the first in vivo, real-time evidence that two unique populations of CA1 cells encode trace conditioning and extinction learning, two distinct phases of hippocampal-dependent learning. The population of cells responsible for the representation of extinction learning emerges within one session of extinction training. Second, we perform calcium imaging in a mouse model containing a total knockout of NEXMIF, a gene causative of autism spectrum disorder. We reveal that loss of NEXMIF causes over-synchronization of the CA1 circuit, particularly during locomotion, impairing the information encoding capacity of the network. Finally, we conduct voltage imaging of CA1 pyramidal cells and parvalbumin (PV)-positive interneurons, with simultaneous recording of local field potential (LFP), to characterize how cellular-level membrane dynamics and spiking relate to network-level LFP. We demonstrate that in PV neurons, membrane potential oscillations in the theta frequency range show consistent synchrony with LFP theta oscillations and organize spike timing of the PV population relative to LFP theta, indicating that PV interneurons orchestrate theta rhythmicity in the CA1 network. In summary, this dissertation utilizes genetically-encoded optical reporters of neural activity, providing critical insights into the function of the CA1 as a flexible, diverse network of individual neurons.

Dans le cervelet, l’interaction entre l’activité des fibres parallèles (FPs) et celle de la fibre grimpante (FG), les deux principaux inputs excitateurs des neurones de Purkinje (NPs), engendre des signaux calciques supra-linéaires procurant des informations sur leur activité concomitante. Ce phénomène déclenche des mécanismes synaptiques qui induisent la dépression à court- ou à long-terme des synapses FP-NPs. L’activation des FPs génère des signaux calciques locaux confinés aux épines activées tandis que l’activation de la FG génère une dépolarisation qui se propage passivement dans les dendrites. Cette dépolarisation transitoire qui n’est pas locale joue un rôle important dans la régulation de la signalisation locale des FPs et leur plasticité. L’étude menée durant ma thèse s’est focalisée sur deux principaux points : les canaux dendritiques des NPs activés par la dépolarisation transitoire générée par l’activation de la FG et les mécanismes responsables de la génération des signaux dendritiques supra-linéaires associés à l’activité concomitante des FPs et de la FG. Les résultats reportés ici ont été obtenus en utilisant des méthodes optiques récemment développés.Nous avons caractérisé le comportement des canaux ioniques dendritiques qui sont activés par la dépolarisation dendritique générés par l’activation de la FG. Nous avons découvert que deux différents groupes de canaux ioniques sont sélectivement activés selon le potentiel membranaire initial. En effet, quand les dendrites sont hyperpolarisées, les CF-EPSPs activent principalement des canaux calciques voltage-dépendant (CCVDs) de type T, des canaux SK et des canaux potassiques voltage-dépendant (CPVDs) de type A. Ces derniers maintiennent le potentiel membranaire en dessous de ~0 mV. En revanche, quand les dendrites sont dépolarisées, les CCVDs de type T et les CPVDs de type A s’inactivent complètement et les CF-EPSPs activent des CCVDs de type P/Q, des CPVDs et des canaux BK. L’activation de cet ensemble de canaux déclenche des spikes calciques. Notamment, nous avons établi l’importance des CPVDs de type A dans le control du deuxième ensemble de canaux. En effet, ils limitent l’activation des CCVDs de type P/Q et les canaux potassiques associés empêchant le déclenchement des spikes calciques.Nous avons démontré que l’activation occurrente de la FG et des FPs induit deux différents types de signaux calciques supra-linéaires. L’induction de l’un ou de l’autre dépend du temps entre l’activation des deux inputs, qui est aussi un principal déterminant des mécanismes impliqués dans la génération des ces signaux calciques. Nous avons trouvé que quand les CF-EPSPs se produisent à de courts délais après la fin du burst des FPs, les signaux calciques supra-linéaires associés sont indépendant de l’activation des mGluR1 et sont générés par l’effet combiné de deux mécanismes : l’augmentation du flux calcique via les CCVD de type P/Q activés par la dépolarisation membranaire médiée par l’activation de FPs inactivant les CPVDs de type A; et la saturation transitoire des buffers calciques endogènes durant le burst des PF-EPSPs amplifiant les concentrations du Ca2+ libre. Quand les CF-EPSPs se produisent à de longs délais après la fin du burst des PF-EPSPs, les signaux calciques supra-linéaires associés dépendent de l’activation des mGluR1 et n’impliquent aucun des mécanismes précédents. Dans ce cas, nous avons démontré que les signaux calciques supra-linéaires sont corrélés avec l’augmentation du flux calcique via les conductances cationiques associées à l’activation des mGluR1.Les résultats reportés ici ont avancé notre compréhension sur la génération des signaux calciques supra-linéaires associés à l’activité concomitante des PFs et de la FGs ainsi les mécanismes qui y sont impliqués. Néanmoins, nous n’avons pas pu procurer une réponse définitive sur la nature du flux calcique médiant les signaux calciques supra-linéaires dépendant de l’activation des mGluR1s. / In the cerebellum, the interplay between PFs and CF inputs generates supralinear Ca2+ signals that provide the information on their concomitant occurrance. These phenomena trigger both short- and long-term depression at PF-PN synapses associated with motor learning and coordination, i.e. the primary functions of the cerebellum. While activation of PFs elicits local Ca2+ transients that are confined to activated spines, the activation of the CF generates a large depolarization that spreads passively into the dendrites. The CF-mediated transient dendritic depolarization, not localized, plays a fundamental role in dendritic integration and in regulating local PF signals and their plasticity at distal sites. The study carried out in my thesis addressed two crucial questions of this problem: the dendritic ion channels activated by the CF-mediated dendritic depolarization at different initial Vm and the mechanisms underlying dendritic supralinear Ca2+ signals associated with concomitant PF and CF activity. The results reported here were obtained using recent optical methods of Vm imaging and ultrafast Ca2+ imaging with low affinity Ca2+ and high affinity Ca2+ indicators combined with pharmacological analysis.During the first part of my work, I characterized the behavior of the dendritic Ca2+ and K+ channels activated by CF-EPSPs at different initial dendritic Vm, using optical measurements of Vm and Ca2+ transients. We found that two different sets of ion channels are selectively activated at different states. When the dendrite is hyperpolarized, CF-EPSPs mainly activate T-type voltage-gated Ca2+ channels (VGCCs), SK channels and A-type voltage-gated Ca2+ channels (VGKCs) that limit the transient Vm below ~0 mV. When in contrast the dendrite is depolarized, T-type VGCCs and A-type VGKCs are inactivated and CF-EPSPs activate P/Q-type VGCCs, high-voltage activated VGKCs and BK channels, initiating Ca2+ spikes. We demonstrated that A-type VGKCs play a crucial role in controlling the second set of channels. Indeed, these channels limit the activation of P/Q-type VGCCs and associated K+ channels, preventing Ca2+ spikes.During the second part of my work, we demonstrated that the concomitant activation of PF and CF triggers two different types of supralinear Ca2+ signals. The activation of one or the other path depends on the delay between the activation of the two inputs which is the crucial discriminator of the mechanisms involved in the generation of supralinear Ca2+ signals. We found that when CF-EPSPs occur near the end of a burst of PFs, the associated supralinear Ca2+ transients are independent of the activation of mGluR1 and are produced by a combined effect of two mechanisms: the increased Ca2+ influx through P/Q-type VGCCs enabled by PF-depolarization inactivating A-type VGKCs; and the transient saturation of endogenous Ca2+ buffers during the PF-EPSP burst amplifying free Ca2+ concentration. When CF-EPSPs occur at longer delays after the end of the PF burst, the associated supralinear Ca2+ transients are mGluR1-dependent and do not involve the mechanisms underlying the generation of mGluR1-independent supralinear Ca2+ transients. Instead, an entirely different mechanism is recruited. We found that, the supralinear Ca2+ transients were correlated with an increase in mGluR1-dependent Ca2+ influx via the slow mGluR1-activated cation conductance.The results reported here advance our understanding of the generation of supralinear Ca2+ transients associated with the concomitant PF and CF activity with respect to the potential molecular mechanisms that are involved. Nevertheless, we were not able to provide a definitive answer on the nature of the Ca2+ influx mediating mGluR1-depedent supralinear Ca2+ signals. This issue must be further explored in future experiments.

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