Plume characteristics and dynamics of UV and IR laser-desorbed oligonucleotides

Laser desorption of dye-tagged oligonucleotides was studied using laser-induced fluorescence imaging. Desorption with ultra violet (UV) and infra-red (IR) lasers resulted in forward directed plumes of molecules. In the case of UV desorption, the initial shot desorbed approximately seven-fold more material than subsequent shots. In contrast, the initial shot in IR desorption resulted in the ejection of less material compared to subsequent shots and these plumes had a component directed along the path of the laser. Thermal equilibrium of the molecules in the plume was achieved after approximately 25 µs with a spread in molecular temperature which was described by a modified Maxwell-Boltzmann equation.

Development of a novel mass spectrometric technique for studying DNA damage

An experimental system, based upon UV and IR laser desorption, has been constructed to enable the production and characterization of neutral biomolecular targets. These targets are to be used for interaction experiments investigating radiation-induced damage to DNA. The viability of the laser-desorption techniques of MALDI (matrix-assisted laser-desorption ionization), SALDI (surface-assisted laser-desorption ionization) and DIOS (desorption/ionization on silicon), for production of these gas targets is discussed in the present paper. Fluorescent dye tagging and LIF (laser-induced fluorescence) imaging has been used to characterize the biomolecular plumes, revealing their spatial density profiles and temporal evolution. © The Authors Journal compilation. © 2009 Biochemical Society.

Development of human central nervous system 3D in vitro models for preclinical research

Neurological disorders are a major concern in modern societies, with increasing prevalence mainly related with the higher life expectancy. Most of the current available therapeutic options can only control and ameliorate the patients’ symptoms, often be-coming refractory over time. Therapeutic breakthroughs and advances have been hampered by the lack of accurate central nervous system (CNS) models. The develop-ment of these models allows the study of the disease onset/progression mechanisms and the preclinical evaluation of novel therapeutics. This has traditionally relied on genetically engineered animal models that often diverge considerably from the human phenotype (developmentally, anatomically and physiologically) and 2D in vitro cell models, which fail to recapitulate the characteristics of the target tissue (cell-cell and cell-matrix interactions, cell polarity). The in vitro recapitulation of CNS phenotypic and functional features requires the implementation of advanced culture strategies that enable to mimic the in vivo struc-tural and molecular complexity. Models based on differentiation of human neural stem cells (hNSC) in 3D cultures have great potential as complementary tools in preclinical research, bridging the gap between human clinical studies and animal models. This thesis aimed at the development of novel human 3D in vitro CNS models by integrat-ing agitation-based culture systems and a wide array of characterization tools. Neural differentiation of hNSC as 3D neurospheres was explored in Chapter 2. Here, it was demonstrated that human midbrain-derived neural progenitor cells from fetal origin (hmNPC) can generate complex tissue-like structures containing functional dopaminergic neurons, as well as astrocytes and oligodendrocytes. Chapter 3 focused on the development of cellular characterization assays for cell aggregates based on light-sheet fluorescence imaging systems, which resulted in increased spatial resolu-tion both for fixed samples or live imaging. The applicability of the developed human 3D cell model for preclinical research was explored in Chapter 4, evaluating the poten-tial of a viral vector candidate for gene therapy. The efficacy and safety of helper-dependent CAV-2 (hd-CAV-2) for gene delivery in human neurons was evaluated, demonstrating increased neuronal tropism, efficient transgene expression and minimal toxicity. The potential of human 3D in vitro CNS models to mimic brain functions was further addressed in Chapter 5. Exploring the use of 13C-labeled substrates and Nucle-ar Magnetic Resonance (NMR) spectroscopy tools, neural metabolic signatures were evaluated showing lineage-specific metabolic specialization and establishment of neu-ron-astrocytic shuttles upon differentiation. Chapter 6 focused on transferring the knowledge and strategies described in the previous chapters for the implementation of a scalable and robust process for the 3D differentiation of hNSC derived from human induced pluripotent stem cells (hiPSC). Here, software-controlled perfusion stirred-tank bioreactors were used as technological system to sustain cell aggregation and dif-ferentiation. The work developed in this thesis provides practical and versatile new in vitro ap-proaches to model the human brain. Furthermore, the culture strategies described herein can be further extended to other sources of neural phenotypes, including pa-tient-derived hiPSC. The combination of this 3D culture strategy with the implemented characterization methods represents a powerful complementary tool applicable in the drug discovery, toxicology and disease modeling.

Etude des propriétés dynamiques de la sécrétion régulée des vésicules glutamatergiques des astrocytes

RESUME GRAND PUBLICLe cerveau est composé de différents types cellulaires, dont les neurones et les astrocytes. Faute de moyens pour les observer, les astrocytes sont très longtemps restés dans l'ombre alors que les neurones, bénéficiant des outils ad hoc pour être stimulés et étudiés, ont fait l'objet de toutes les attentions. Le développement de l'imagerie cellulaire et des outils fluorescents ont permis d'observer ces cellules non électriquement excitables et d'obtenir des informations qui laissent penser que ces cellules sont loin d'être passives et participent activement au fonctionnement cérébral. Cette participation au fonctionnement cérébral se fait en partie par le biais de la libération de substances neuro-actives (appellées gliotransmetteurs) que les astrocytes libèrent à proximité des synapses permettant ainsi de moduler le fonctionnement neuronal. Cette libération de gliotransmetteurs est principalement causée par l'activité neuronale que les astrocytes sont capables de sentir. Néanmoins, nous savons encore peu de chose sur les propriétés précises de la libération des gliotransmetteurs. Comprendre les propriétés spatio-temporelles de cette libération est essentiel pour comprendre le mode de communication de ces cellules et leur implication dans la transmission de l'information cérébrale. En utilisant des outils fluorescents récemment développés et en combinant différentes techniques d'imagerie cellulaire, nous avons pu obtenir des informations très précises sur la libération de ces gliotransmetteurs par les astrocytes. Nous avons ainsi confirmé que cette libération était un processus très rapide et qu'elle était contrôlée par des augmentations de calcium locales et rapides. Nous avons également décrit une organisation complexe de la machinerie supportant la libération des gliotransmetteurs. Cette organisation complexe semble être à la base de la libération extrêmement rapide des gliotransmetteurs. Cette rapidité de libération et cette complexité structurelle semblent indiquer que les astrocytes sont des cellules particulièrement adaptées à une communication rapide et qu'elles peuvent, au même titre que les neurones dont elles seraient les partenaires légitimes, participer à la transmission et à l'intégration de l'information cérébrale.RESUMEDe petites vésicules, les « SLMVs » ou « Synaptic Like MicroVesicles », exprimant des transporteurs vésiculaires du glutamate (VGluTs) et libérant du glutamate par exocytose régulée, ont récemment été décrites dans les astrocytes en culture et in situ. Néanmoins, nous savons peu de chose sur les propriétés précises de la sécrétion de ces SLMVs. Contrairement aux neurones, le couplage stimulussécrétion des astrocytes n'est pas basé sur l'ouverture des canaux calciques membranaires mais nécessite l'intervention de seconds messagers et la libération du calcium par le reticulum endoplasmique (RE). Comprendre les propriétés spatio-temporelles de la sécrétion astrocytaire est essentiel pour comprendre le mode de communication de ces cellules et leur implication dans la transmission de l'information cérébrale. Nous avons utilisé des outils fluorescents récemment développés pour étudier le recyclage des vésicules synaptiques glutamatergiques comme les colorants styryles et la pHluorin afin de pouvoir suivre la sécrétion des SLMVs à l'échelle de la cellule mais également à l'échelle des évènements. L'utilisation combinée de l'épifluorescence et de la fluorescence à onde évanescente nous a permis d'obtenir une résolution temporelle et spatiale sans précédent. Ainsi avons-nous confirmé que la sécrétion régulée des astrocytes était un processus très rapide (de l'ordre de quelques centaines de millisecondes). Nous avons découvert que cette sécrétion est contrôlée par des augmentations de calcium locales et rapides. Nous avons également décrit des compartiments cytosoliques délimités par le RE à proximité de la membrane plasmique et contenant les SLMVs. Cette organisation semble être à la base du couplage rapide entre l'activation des GPCRs et la sécrétion. L'existence de compartiments subcellulaires indépendants permettant de contenir les messagers intracellulaires et de limiter leur diffusion semble compenser de manière efficace la nonexcitabilité électrique des astrocytes. Par ailleurs, l'existence des différents pools de vésicules recrutés séquentiellement et fusionnant selon des modalités distinctes ainsi que l'existence de mécanismes permettant le renouvellement de ces pools lors de la stimulation suggèrent que les astrocytes peuvent faire face à une stimulation soutenue de leur sécrétion. Ces données suggèrent que la libération de gliotransmetteurs par exocytose régulée n'est pas seulement une propriété des astrocytes en culture mais bien le résultat d'une forte spécialisation de ces cellules pour la sécrétion. La rapidité de cette sécrétion donne aux astrocytes toutes les compétences pour pouvoir intervenir de manière active dans la transmission et l'intégration de l'information.ABSTRACTRecently, astrocytic synaptic like microvesicles (SLMVs), that express vesicular glutamate transporters (VGluTs) and are able to release glutamate by Ca2+-dependent regulated exocytosis, have been described both in tissue and in cultured astrocytes. Nevertheless, little is known about the specific properties of regulated secretion in astrocytes. Important differences may exist between astrocytic and neuronal exocytosis, starting from the fact that stimulus-secretion coupling in astrocytes is voltage independent, mediated by G-protein-coupled receptors and the release of Ca2+ from internal stores. Elucidating the spatiotemporal properties of astrocytic exo-endocytosis is, therefore, of primary importance for understanding the mode of communication of these cells and their role in brain signaling. We took advantage of fluorescent tools recently developed for studying recycling of glutamatergic vesicles at synapses like styryl dyes and pHluorin in order to follow exocytosis and endocytosis of SLMVs at the level of the entire cell or at the level of single event. We combined epifluorescence and total internal reflection fluorescence imaging to investigate, with unprecedented temporal and spatial resolution, the events underlying the stimulus-secretion in astrocytes. We confirmed that exo-endocytosis process in astrocytes proceeds with a time course on the millisecond time scale. We discovered that SLMVs exocytosis is controlled by local and fast Ca2+ elevations; indeed submicrometer cytosolic compartments delimited by endoplasmic reticulum (ER) tubuli reaching beneath the plasma membrane and containing SLMVs. Such complex organization seems to support the fast stimulus-secretion coupling reported here. Independent subcellular compartments formed by ER, SLMVs and plasma membrane containing intracellular messengers and limiting their diffusion seem to compensate efficiently the non-electrical excitability of astrocytes. Moreover, the existence of two pools of SLMVs which are sequentially recruited suggests a compensatory mechanisms allowing the refill of SLMVs and supporting exocytosis process over a wide range of multiple stimuli. These data suggest that regulated secretion is not only a feature of cultured astrocytes but results from a strong specialization of these cells. The rapidity of secretion demonstrates that astrocytes are able to actively participate in brain information transmission and processing.

Fast subplasma membrane Ca2+ transients control exo-endocytosis of synaptic-like microvesicles in astrocytes

Astrocytes are the most abundant glial cell type in the brain. Although not apposite for long-range rapid electrical communication, astrocytes share with neurons the capacity of chemical signaling via Ca(2+)-dependent transmitter exocytosis. Despite this recent finding, little is known about the specific properties of regulated secretion and vesicle recycling in astrocytes. Important differences may exist with the neuronal exocytosis, starting from the fact that stimulus-secretion coupling in astrocytes is voltage independent, mediated by G-protein-coupled receptors and the release of Ca(2+) from internal stores. Elucidating the spatiotemporal properties of astrocytic exo-endocytosis is, therefore, of primary importance for understanding the mode of communication of these cells and their role in brain signaling. We here take advantage of fluorescent tools recently developed for studying recycling of glutamatergic vesicles at synapses (Voglmaier et al., 2006; Balaji and Ryan, 2007); we combine epifluorescence and total internal reflection fluorescence imaging to investigate with unprecedented temporal and spatial resolution, the stimulus-secretion coupling underlying exo-endocytosis of glutamatergic synaptic-like microvesicles (SLMVs) in astrocytes. Our main findings indicate that (1) exo-endocytosis in astrocytes proceeds with a time course on the millisecond time scale (tau(exocytosis) = 0.24 +/- 0.017 s; tau(endocytosis) = 0.26 +/- 0.03 s) and (2) exocytosis is controlled by local Ca(2+) microdomains. We identified submicrometer cytosolic compartments delimited by endoplasmic reticulum tubuli reaching beneath the plasma membrane and containing SLMVs at which fast (time-to-peak, approximately 50 ms) Ca(2+) events occurred in precise spatial-temporal correlation with exocytic fusion events. Overall, the above characteristics of transmitter exocytosis from astrocytes support a role of this process in fast synaptic modulation.

Acute insult of ammonia leads to calcium-dependent glutamate release from cultured astrocytes, an effect of pH.

Hyperammonemia is a key factor in the pathogenesis of hepatic encephalopathy (HE) as well as other metabolic encephalopathies, such as those associated with inherited disorders of urea cycle enzymes and in Reye's syndrome. Acute HE results in increased brain ammonia (up to 5 mM), astrocytic swelling, and altered glutamatergic function. In the present study, using fluorescence imaging techniques, acute exposure (10 min) of ammonia (NH4+/NH3) to cultured astrocytes resulted in a concentration-dependent, transient increase in [Ca2+]i. This calcium transient was due to release from intracellular calcium stores, since the response was thapsigargin-sensitive and was still observed in calcium-free buffer. Using an enzyme-linked fluorescence assay, glutamate release was measured indirectly via the production of NADH (a naturally fluorescent product when excited with UV light). NH4+/NH3 (5 mM) stimulated a calcium-dependent glutamate release from cultured astrocytes, which was inhibited after preincubation with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester but unaffected after preincubation with glutamate transport inhibitors dihydrokainate and DL-threo-beta-benzyloxyaspartate. NH4+/NH3 (5 mM) also induced a transient intracellular alkaline shift. To investigate whether the effects of NH4+/NH3 were mediated by an increase in pH(i), we applied trimethylamine (TMA+/TMA) as another weak base. TMA+/TMA (5 mM) induced a similar transient increase in both pH(i) and [Ca2+]i (mobilization from intracellular calcium stores) and resulted in calcium-dependent release of glutamate. These results indicate that an acute exposure to ammonia, resulting in cytosolic alkalinization, leads to calcium-dependent glutamate release from astrocytes. A deregulation of glutamate release from astrocytes by ammonia could contribute to glutamate dysfunction consistently observed in acute HE.

The Central Nervous System as Target for Antihypertensive Actions of a Proline-Rich Peptide from Bothrops jararaca Venom

Pyroglutamyl proline-rich oligopeptides, present in the venom of the pit viper Bothrops jararaca (Bj-PROs), are the first described naturally occurring inhibitors of the angiotensin I-converting enzyme (ACE). The inhibition of ACE by the decapeptide Bj-PRO-10c (imaging by confocal microscopy and fluorescence imaging plate reader analysis, we have characterized Bj-PRO-10c-induced [Ca(2+)](i) transients in rat brain cells as being independent from bradykinin-mediated effects and ACE inhibition. Bj-PRO-10c induced pertussis toxin-sensitive G(i/o)-protein activity mediated through a yet unknown receptor, influx and liberation of calcium from intracellular stores, as well as reduction of intracellular cAMP levels. Bj-PRO-10c promoted glutamate and GABA release that may be responsible for its antihypertensive activity and its effect on HR. (C) 2010 International Society for Advancement of Cytometry

Cryo-electron microscopy and single molecule fluorescent microscopy detect CD4 receptor induced HIV size expansion prior to cell entry

Viruses are often thought to have static structure, and they only remodel after the viruses have entered target cells. Here, we detected a size expansion of virus particles prior to viral entry using cryo-electron microscopy (cryo-EM) and single molecule fluorescence imaging. HIV expanded both under cell-free conditions with soluble receptor CD4 (sCD4) targeting the CD4 binding site on the HIV-1 envelope protein (Env) and when HIV binds to receptor on cellular membrane. We have shown that the HIV Env is needed to facilitate receptor induced virus size expansions, showing that the 'lynchpin' for size expansion is highly specific. We demonstrate that the size expansion required maturation of HIV and an internal capsid core with wild type stability, suggesting that different HIV compartments are linked and are involved in remodelling. Our work reveals a previously unknown event in HIV entry, and we propose that this pre-entry priming process enables HIV particles to facilitate the subsequent steps in infection.

Assessment of ALA-induced PpIX production in porcine skin pretreated with microneedles

Photodynamic therapy (PDT) is used for skin treatments of premalignant and cancer lesions and recognized as a non-invasive technique that combines tissue photosensitization and subsequent exposure to light to induce cell death. However, it is limited to the treatment of superficial lesions, mainly due to the low cream penetration. Therefore, the improvement of transdermal distribution of aminolevulinic acid (ALA) is needed. In this study, the kinetics and homogeneity of production of ALA-induced PpIX after the skin pre-treatment with microneedles rollers of 0.5, 1.0 and 1.5 mm length were investigated. An improvement in homogeneity and production of PpIX was shown in a porcine model. Widefield fluorescence imaging three hours after the topical application of ALA-cream in the combined treatment with microeedles rollers.

Surface-plasmon optical and electrochemical characterization of biofunctional surface architectures

The development and characterization of biomolecule sensor formats based on the optical technique Surface Plasmon Resonance (SPR) Spectroscopy and electrochemical methods were investigated. The study can be divided into two parts of different scope. In the first part new novel detection schemes for labeled targets were developed on the basis of the investigations in Surface-plamon Field Enhanced Spectroscopy (SPFS). The first one is SPR fluorescence imaging formats, Surface-plamon Field Enhanced Fluorescence Microscopy (SPFM). Patterned self assembled monolayers (SAMs) were prepared and used to direct the spatial distribution of biomolecules immobilized on surfaces. Here the patterned monolayers would serve as molecular templates to secure different biomolecules to known locations on a surface. The binding processed of labeled target biomolecules from solution to sensor surface were visually and kinetically recorded by the fluorescence microscope, in which fluorescence was excited by the evanescent field of propagating plasmon surface polaritons. The second format which also originates from SPFS technique, Surface-plamon Field Enhanced Fluorescence Spectrometry (SPFSm), concerns the coupling of a fluorometry to normal SPR setup. A spectrograph mounted in place of photomultiplier or microscope can provide the information of fluorescence spectrum as well as fluorescence intensity. This study also firstly demonstrated the analytical combination of surface plasmon enhanced fluorescence detection with analyte tagged by semiconducting nano- crystals (QDs). Electrochemically addressable fabrication of DNA biosensor arrays in aqueous environment was also developed. An electrochemical method was introduced for the directed in-situ assembly of various specific oligonucleotide catcher probes onto different sensing elements of a multi-electrode array in the aqueous environment of a flow cell. Surface plasmon microscopy (SPM) is utilized for the on-line recording of the various functionalization steps. Hybridization reactions between targets from solution to the different surface-bound complementary probes are monitored by surface-plasmon field-enhanced fluorescence microscopy (SPFM) using targets that are either labeled with organic dyes or with semiconducting quantum dots for color-multiplexing. This study provides a new approach for the fabrication of (small) DNA arrays and the recording and quantitative evaluation of parallel hybridization reactions. In the second part of this work, the ideas of combining the SP optical and electrochemical characterization were extended to tethered bilayer lipid membrane (tBLM) format. Tethered bilayer lipid membranes provide a versatile model platform for the study of many membrane related processes. The thiolipids were firstly self-assembled on ultraflat gold substrates. Fusion of the monolayers with small unilamellar vesicles (SUVs) formed the distal layer and the membranes thus obtained have the sealing properties comparable to those of natural membranes. The fusion could be monitored optically by SPR as an increase in reflectivity (thickness) upon formation of the outer leaflet of the bilayer. With EIS, a drop in capacitance and a steady increase in resistance could be observed leading to a tightly sealing membrane with low leakage currents. The assembly of tBLMs and the subsequent incorporation of membrane proteins were investigated with respect to their potential use as a biosensing system. In the case of valinomycin the potassium transport mediated by the ion carrier could be shown by a decrease in resistance upon increasing potassium concentration. Potential mediation of membrane pores could be shown for the ion channel forming peptide alamethicin (Alm). It was shown that at high positive dc bias (cis negative) Alm channels stay at relatively low conductance levels and show higher permeability to potassium than to tetramethylammonium. The addition of inhibitor amiloride can partially block the Alm channels and results in increase of membrane resistance. tBLMs are robust and versatile model membrane architectures that can mimic certain properties of biological membranes. tBLMs with incorporated lipopolysaccharide (LPS) and lipid A mimicking bacteria membranes were used to probe the interactions of antibodies against LPS and to investigate the binding and incorporation of the small antimicrobial peptide V4. The influence of membrane composition and charge on the behavior of V4 was also probed. This study displays the possibility of using tBLM platform to record and valuate the efficiency or potency of numerous synthesized antimicrobial peptides as potential drug candidates.

Mapping the elastic response of epithelial apical cell membranes suspended across porous array

As the elastic response of cell membranes to mechanical stimuli plays a key role in various cellular processes, novel biophysical strategies to quantify the elasticity of native membranes under physiological conditions at a nanometer scale are gaining interest. In order to investigate the elastic response of apical membranes, elasticity maps of native membrane sheets, isolated from MDCK II (Madine Darby Canine kidney strain II) epithelial cells, were recorded by local indentation with an Atomic Force Microscope (AFM). To exclude the underlying substrate effect on membrane indentation, a highly ordered gold coated porous array with a pore diameter of 1.2 μm was used to support apical membranes. Overlays of fluorescence and AFM images show that intact apical membrane sheets are attached to poly-D-lysine coated porous substrate. Force indentation measurements reveal an extremely soft elastic membrane response if it is indented at the center of the pore in comparison to a hard repulsion on the adjacent rim used to define the exact contact point. A linear dependency of force versus indentation (-dF/dh) up to 100 nm penetration depth enabled us to define an apparent membrane spring constant (kapp) as the slope of a linear fit with a stiffness value of for native apical membrane in PBS. A correlation between fluorescence intensity and kapp is also reported. Time dependent hysteresis observed with native membranes is explained by a viscoelastic solid model of a spring connected to a Kelvin-Voight solid with a time constant of 0.04 s. No hysteresis was reported with chemically fixated membranes. A combined linear and non linear elastic response is suggested to relate the experimental data of force indentation curves to the elastic modulus and the membrane thickness. Membrane bending is the dominant contributor to linear elastic indentation at low loads, whereas stretching is the dominant contributor for non linear elastic response at higher loads. The membrane elastic response was controlled either by stiffening with chemical fixatives or by softening with F-actin disrupters. Overall, the presented setup is ideally suitable to study the interactions of the apical membrane with the underlying cytoskeleton by means of force indentation elasticity maps combined with fluorescence imaging.

Inhibition of the human epithelial calcium channel TRPV6 by 2-aminoethoxydiphenyl borate (2-APB)

TRPV6, a highly calcium-selective member of the transient receptor potential (TRP) channel superfamily, is a major pathway for calcium absorption in the fetal and adult body. It is expressed abundantly in the duodenum, the placenta and exocrine tissues. TRVP6 was postulated to contribute to store-operated calcium channel (SOC) activity in certain cell types such as exocrine cells. In this study, we tested 2-APB, a widely used SOC inhibitor on human TRPV6 (hTRPV6) activity using fluorescence imaging, patch clamp and radioactive tracer techniques in transiently and stably transfected HEK293 cells. We found that the basal calcium and cadmium influx was higher in HEK293 cells transfected with hTRPV6 than in non-transfected cells. 2-APB inhibited hTRPV6 activity in both transient and stably transfected cells. This effect was slightly sensitive toward extracellular calcium. The extracellular sodium concentration did not affect the inhibition of hTRPV6 by 2-APB. However, N-methyl-d-glucamine significantly diminished the inhibitory effect of 2-APB presumably through direct interaction with this compound. Furthermore, 2-APB inhibited the activity of TRPV6 orthologs but not human TRPV5. 2-APB may serve as a parental compound for the development of therapeutic strategies specifically targeting the hTRPV6 calcium channel.

Self-organizing cultured neural networks: Image analysis techniques for longitudinal tracking and modeling of the underlying network structure

Esta tesis estudia la evolución estructural de conjuntos de neuronas como la capacidad de auto-organización desde conjuntos de neuronas separadas hasta que forman una red (clusterizada) compleja. Esta tesis contribuye con el diseño e implementación de un algoritmo no supervisado de segmentación basado en grafos con un coste computacional muy bajo. Este algoritmo proporciona de forma automática la estructura completa de la red a partir de imágenes de cultivos neuronales tomadas con microscopios de fase con una resolución muy alta. La estructura de la red es representada mediante un objeto matemático (matriz) cuyos nodos representan a las neuronas o grupos de neuronas y los enlaces son las conexiones reconstruidas entre ellos. Este algoritmo extrae también otras medidas morfológicas importantes que caracterizan a las neuronas y a las neuritas. A diferencia de otros algoritmos hasta el momento, que necesitan de fluorescencia y técnicas inmunocitoquímicas, el algoritmo propuesto permite el estudio longitudinal de forma no invasiva posibilitando el estudio durante la formación de un cultivo. Además, esta tesis, estudia de forma sistemática un grupo de variables topológicas que garantizan la posibilidad de cuantificar e investigar la progresión de las características principales durante el proceso de auto-organización del cultivo. Nuestros resultados muestran la existencia de un estado concreto correspondiente a redes con configuracin small-world y la emergencia de propiedades a micro- y meso-escala de la estructura de la red. Finalmente, identificamos los procesos físicos principales que guían las transformaciones morfológicas de los cultivos y proponemos un modelo de crecimiento de red que reproduce el comportamiento cuantitativamente de las observaciones experimentales. ABSTRACT The thesis analyzes the morphological evolution of assemblies of living neurons, as they self-organize from collections of separated cells into elaborated, clustered, networks. In particular, it contributes with the design and implementation of a graph-based unsupervised segmentation algorithm, having an associated very low computational cost. The processing automatically retrieves the whole network structure from large scale phase-contrast images taken at high resolution throughout the entire life of a cultured neuronal network. The network structure is represented by a mathematical object (a matrix) in which nodes are identified neurons or neurons clusters, and links are the reconstructed connections between them. The algorithm is also able to extract any other relevant morphological information characterizing neurons and neurites. More importantly, and at variance with other segmentation methods that require fluorescence imaging from immunocyto- chemistry techniques, our measures are non invasive and entitle us to carry out a fully longitudinal analysis during the maturation of a single culture. In turn, a systematic statistical analysis of a group of topological observables grants us the possibility of quantifying and tracking the progression of the main networks characteristics during the self-organization process of the culture. Our results point to the existence of a particular state corresponding to a small-world network configuration, in which several relevant graphs micro- and meso-scale properties emerge. Finally, we identify the main physical processes taking place during the cultures morphological transformations, and embed them into a simplified growth model that quantitatively reproduces the overall set of experimental observations.

Self-organizing cultured neural networks: Image analysis techniques for longitudinal tracking and modeling of the underlying network structure (versión actualizada)

Esta tesis estudia la evolución estructural de conjuntos de neuronas como la capacidad de auto-organización desde conjuntos de neuronas separadas hasta que forman una red (clusterizada) compleja. Esta tesis contribuye con el diseño e implementación de un algoritmo no supervisado de segmentación basado en grafos con un coste computacional muy bajo. Este algoritmo proporciona de forma automática la estructura completa de la red a partir de imágenes de cultivos neuronales tomadas con microscopios de fase con una resolución muy alta. La estructura de la red es representada mediante un objeto matemático (matriz) cuyos nodos representan a las neuronas o grupos de neuronas y los enlaces son las conexiones reconstruidas entre ellos. Este algoritmo extrae también otras medidas morfológicas importantes que caracterizan a las neuronas y a las neuritas. A diferencia de otros algoritmos hasta el momento, que necesitan de fluorescencia y técnicas inmunocitoquímicas, el algoritmo propuesto permite el estudio longitudinal de forma no invasiva posibilitando el estudio durante la formación de un cultivo. Además, esta tesis, estudia de forma sistemática un grupo de variables topológicas que garantizan la posibilidad de cuantificar e investigar la progresión de las características principales durante el proceso de auto-organización del cultivo. Nuestros resultados muestran la existencia de un estado concreto correspondiente a redes con configuracin small-world y la emergencia de propiedades a micro- y meso-escala de la estructura de la red. Finalmente, identificamos los procesos físicos principales que guían las transformaciones morfológicas de los cultivos y proponemos un modelo de crecimiento de red que reproduce el comportamiento cuantitativamente de las observaciones experimentales. ABSTRACT The thesis analyzes the morphological evolution of assemblies of living neurons, as they self-organize from collections of separated cells into elaborated, clustered, networks. In particular, it contributes with the design and implementation of a graph-based unsupervised segmentation algorithm, having an associated very low computational cost. The processing automatically retrieves the whole network structure from large scale phase-contrast images taken at high resolution throughout the entire life of a cultured neuronal network. The network structure is represented by a mathematical object (a matrix) in which nodes are identified neurons or neurons clusters, and links are the reconstructed connections between them. The algorithm is also able to extract any other relevant morphological information characterizing neurons and neurites. More importantly, and at variance with other segmentation methods that require fluorescence imaging from immunocyto- chemistry techniques, our measures are non invasive and entitle us to carry out a fully longitudinal analysis during the maturation of a single culture. In turn, a systematic statistical analysis of a group of topological observables grants us the possibility of quantifying and tracking the progression of the main networks characteristics during the self-organization process of the culture. Our results point to the existence of a particular state corresponding to a small-world network configuration, in which several relevant graphs micro- and meso-scale properties emerge. Finally, we identify the main physical processes taking place during the cultures morphological transformations, and embed them into a simplified growth model that quantitatively reproduces the overall set of experimental observations.

Calcium electrogenesis in distal apical dendrites of layer 5 pyramidal cells at a critical frequency of back-propagating action potentials

Action potentials in juvenile and adult rat layer-5 neocortical pyramidal neurons can be initiated at both axonal and distal sites of the apical dendrite. However, little is known about the interaction between these two initiation sites. Here, we report that layer 5 pyramidal neurons are very sensitive to a critical frequency of back-propagating action potentials varying between 60 and 200 Hz in different neurons. Bursts of four to five back-propagating action potentials above the critical frequency elicited large regenerative potentials in the distal dendritic initiation zone. The critical frequency had a very narrow range (10–20 Hz), and the dendritic regenerative activity led to further depolarization at the soma. The dendritic frequency sensitivity was suppressed by blockers of voltage-gated calcium channels, and also by synaptically mediated inhibition. Calcium-fluorescence imaging revealed that the site of largest transient increase in intracellular calcium above the critical frequency was located 400–700 μm from the soma at the site for initiation of calcium action potentials. Thus, the distal dendritic initiation zone can interact with the axonal initiation zone, even when inputs to the neuron are restricted to regions close to the soma, if the output of the neuron exceeds a critical frequency.