Determining the neurotransmitter concentration profile at active synapses

Establishing the temporal and concentration profiles of neurotransmitters during synaptic release is an essential step towards understanding the basic properties of inter-neuronal communication in the central nervous system. A variety of ingenious attempts has been made to gain insights into this process, but the general inaccessibility of central synapses, intrinsic limitations of the techniques used, and natural variety of different synaptic environments have hindered a comprehensive description of this fundamental phenomenon. Here, we describe a number of experimental and theoretical findings that has been instrumental for advancing our knowledge of various features of neurotransmitter release, as well as newly developed tools that could overcome some limits of traditional pharmacological approaches and bring new impetus to the description of the complex mechanisms of synaptic transmission

Functional and structural substrates of increased dosage of Grik4 gene elucidated using multi-modal MRI

Grik4 is the gene responsible for encoding the high-affinity GluK4 subunit of the kainate receptors. Increased dosage of this subunit in the forebrain was linked to an increased level of anxiety, lack of social communication, and depression. On the synaptic level, abnormal synaptic transmission was also reported. The manifestations of this abnormal expression have not been investigated at the circuit level, nor the correlations between those circuits and the abnormal patterns of the behavior previously reported. In this line of work, we aspired to use different non-invasive magnetic resonance imaging (MRI) modalities to elucidate any disturbance that might stem from the increased dosage of Grik4 and how those changes might explain the abnormal behaviors. MRI offers a noninvasive way to look into the intact brain in vivo. Resting-state functional MRI casts light on how the brain function at rest on the network level and has the capability to detect any anomalies that might occur within or between those networks. On the microstructural level, the diffusion MRI is concerned with the underlying features of the tissues, using the diffusion of water molecules as a proxy for that end. Moving more macroscopically, using structural scans, voxel-based morphometry can detect subtle differences in the morphology of the different brain structures. We recorded videos of our animals performing two tasks that have long been linked to anxiety, the open field and the plus-maze tests before acquiring structural and functional scans. Lastly, we recorded blood-oxygenationlevel dependent (BOLD) signals in a different set of animals during electrical stimulation of specific white matter tracts in order to investigate how neuronal activity propagates. Our analysis showed a vast spectrum of changes in the transgenic group relative to the animals in the control group. On the resting-state networks level, we observed an increase in the within-network strength spanning different structures such as the hippocampus, some regions of the cortex, and the hypothalamus. The increased internal coherence or strength in the networks contrasted with a significant reduction in between-networks connectivity for some regions such as parts of the cortex and the hypothalamus, suggesting long-range network decorrelation. Supporting this idea, major white matter (WM) tracts, such as the corpus callosum and the hippocampal commissure, suffered from substantial changes compatible with an important reduction in myelination and/or a decrease in the mean axonal diameter. Macrostructurally speaking, the overexpression of GluK4 subunit had a bimodal effect, with expansion in some cortical areas in the transgenic animals accompanied by a shrinkage in the subcortical regions. Upon stimulating the brain with an electrical current, we noticed a difference in activity propagation between the two hemispheres. In transgenic animals, the evoked activity remained more confined to the stimulated hemisphere, again consistent with an impaired long-range connectivity. The structural changes both, at the micro and macro level, were in tight correlation with different aspects of the behavior including markers of anxiety such as the time spent in the open arms vs the closed arms in the plus-maze test and the time spent in the center vs the corners in the open field test. Our findings reveal how the disruption of kainate receptors, or more globally the glutamate receptors, and the abnormal synaptic transmission can translate into brain-wide changes in connectivity and alter the functional equilibrium between macro-and mesoscopic networks. The postsynaptic enhancement previously reported in the transgenic animals was here reflected in the BOLD signal and measured as an increase in the within-network strength. Importantly, the correlations between the structural changes and the behavior help to put the developmental changes and their behavioral ramifications into context. RESUMEN Grik4 es el gen responsable de codificar la subunidad GluK4 de alta afinidad de los receptores de kainato. El aumento de la dosis de esta subunidad en el prosencéfalo se relacionó con un mayor nivel de ansiedad, falta de comunicación social y depresión. A nivel sináptico, también se informó una transmisión sináptica anormal. Las manifestaciones de esta expresión anormal no se han investigado a nivel de circuito, ni las correlaciones entre esos circuitos y los patrones anormales de la conducta previamente informada. En esta línea de trabajo, aspiramos a utilizar diferentes modalidades de imágenes por resonancia magnética (MRI) no invasivas para dilucidar cualquier alteración que pudiera derivarse del aumento de la dosis de Grik4 y cómo esos cambios podrían explicar los comportamientos anormales. La resonancia magnética ofrece una forma no invasiva de observar el cerebro intacto in vivo. La resonancia magnética funcional en estado de reposo arroja luz sobre cómo funciona el cerebro en reposo en el nivel de la red y tiene la capacidad de detectar cualquier anomalía que pueda ocurrir dentro o entre esas redes. En el nivel microestructural, la resonancia magnética de difusión se ocupa de las características subyacentes de los tejidos utilizando la difusión de moléculas de agua como un proxy para ese fin. Moviéndose más macroscópicamente, utilizando escaneos estructurales, la morfometría basada en vóxeles puede detectar diferencias sutiles en la morfología de las diferentes estructuras cerebrales. Grabamos videos de nuestros animales realizando dos tareas que durante mucho tiempo se han relacionado con la ansiedad, el campo abierto y las pruebas de laberinto positivo antes de adquirir escaneos estructurales y funcionales. Por último, registramos señales dependientes del nivel de oxigenación de la sangre (BOLD) en un grupo diferente de animales durante la estimulación eléctrica de tractos específicos de materia blanca para investigar cómo se propaga la actividad neuronal. Nuestro análisis mostró un amplio espectro de cambios en el grupo transgénico en relación con los animales en el grupo de control. En el nivel de las redes de estado de reposo, observamos un aumento en la fuerza dentro de la red que abarca diferentes estructuras como el hipocampo, algunas regiones de la corteza y el hipotálamo. La mayor coherencia interna o fuerza en las redes contrastó con una reducción significativa en la conectividad entre redes para algunas regiones como partes de la corteza y el hipotálamo, lo que sugiere una descorrelación de redes de largo alcance. Apoyando esta idea, los grandes tractos de materia blanca (WM), como el cuerpo calloso y la comisura del hipocampo, sufrieron cambios sustanciales compatibles con una importante reducción de la mielinización y / o una disminución del diámetro axonal medio. Macroestructuralmente hablando, la sobreexpresión de la subunidad GluK4 tuvo un efecto bimodal, con expansión en algunas áreas corticales en los animales transgénicos acompañada de una contracción en las regiones subcorticales. Al estimular el cerebro con una corriente eléctrica, notamos una diferencia en la propagación de la actividad entre las dos hemiesferas. En los animales transgénicos, la actividad evocada permaneció más confinada al hemisferio estimulado, de nuevo consistente con una conectividad de largo alcance deteriorada. Los cambios estructurales, tanto a nivel micro como macro, estaban en estrecha correlación con diferentes aspectos de la conducta, incluidos marcadores de ansiedad como el tiempo pasado con los brazos abiertos frente a los brazos cerrados en la prueba del laberinto positivo y el tiempo pasado en el centro vs las esquinas en la prueba de campo abierto. Nuestros hallazgos revelan cómo la interrupción de los receptores de kainato, o más globalmente los receptores de glutamato, y la transmisión sináptica anormal pueden traducirse en cambios de conectividad en todo el cerebro y alterar el equilibrio funcional entre las redes macro y mesoscópicas. La mejora postsináptica informada anteriormente en los animales transgénicos se reflejó aquí en la señal BOLD y se midió como un aumento en la fuerza dentro de la red. Es importante destacar que las correlaciones entre los cambios estructurales y elcomportamiento ayudan a contextualizar los cambios en el desarrollo y sus ramificaciones conductuales

Local diffusion in the extracellular space of the brain

The brain extracellular space (ECS) is a vast interstitial reticulum of extreme morphological complexity, composed of narrow gaps separated by local expansions, enabling interconnected highways between neural cells. Constituting on average 20% of brain volume, the ECS is key for intercellular communication, and understanding its diffusional properties is of paramount importance for understanding the brain. Within the ECS, neuroactive substances travel predominantly by diffusion, spreading through the interstitial fluid and the extracellular matrix scaffold after being focally released. The nanoscale dimensions of the ECS render it unresolvable by conventional live tissue compatible imaging methods, and historically diffusion of tracers has been used to indirectly infer its structure. Novel nanoscopic imaging techniques now show that the ECS is a highly dynamic compartment, and that diffusivity in the ECS is more heterogeneous than anticipated, with great variability across brain regions and physiological states. Diffusion is defined primarily by the local ECS geometry, and secondarily by the viscosity of the interstitial fluid, including the obstructive and binding properties of the extracellular matrix. ECS volume fraction and tortuosity both strongly determine diffusivity, and each can be independently regulated e.g. through alterations in glial morphology and the extracellular matrix composition. Here we aim to provide an overview of our current understanding of the ECS and its diffusional properties. We highlight emerging technological advances to respectively interrogate and model diffusion through the ECS, and point out how these may contribute in resolving the remaining enigmas of the ECS.The authors acknowledge funding from the Spanish Ministry of Science and Innovation (PID2020-115896RJ-I00, PID2020-113894RB-I00, PCI2022-135040-2), the Basque Government (GIC21/76, GIU21/048), CIBERNED, Human Frontier Science Program (RGP0036/2020) and Aligning Science Across Parkinson's (ASAP-020505) through the Michael J. Fox Foundation for Parkinson's Research (MJFF)

Rapid diffusion in the brain extracellular space - biophysical constraints and physiological implications

Physiological experiments backed by biophysical models have shown that, in central glutamatergic synapses, changes in extracellular diffusivity or glutamate transporter functions exert significant influences on the excitatory transmission. Failures of transporter functions have also been related to neurological disorders. The underlying biophysical mechanisms remain poorly understood. Here, we first combine two‐photon excitation imaging with electrophysiology to estimate the diffusivity of small soluble molecules, such as glutamate in the hippocampal neuropil (area CA1). Next, we adopt time‐resolved fluorescence anisotropy imaging microscopy to establish the previously unknown instantaneous diffusivity of small molecules in the extracellular space. The result indicates that nanometer‐scale diffusivity in the brain extracellular space is 25‐30% slower than that in free medium. Accounting for this retardation may have fundamental consequences for accurate interpretation of diffusion‐limited reactions in the brain. To obtain insight into the mechanisms contributing to the excitatory signal formation, we incorporate these results in a newly developed Monte‐Carlo model of the typical environment of small excitatory synapses including unevenly distributed receptors and transporters. In addition, we build a macroscopic three‐dimensional compartmental model of the hippocampal neuropil based on available experimental data to examine the effect of transporter distribution on the extracellular landscape of glutamate. Monte‐Carlo simulations show to what extent altering diffusivity inside or outside the synaptic cleft affect synaptic responses. Modelling also predicts that extrasynaptic transporters have little effect on fast synaptic transmission through AMPARs and NMDARs. However, they influence the responses of high‐affinity extrasynaptic receptors, such as NMDA or metabotropic receptors. Conversely, intra‐cleft glutamate transporters should significantly attenuate activation of synaptic transmission. On a larger neuropil scale, failure of >95% transporters is required for any significant elevation of glutamate (above 1‐2 μM) to occur. Our data shed light on fundamental biophysical constraints important for a better understanding of excitatory signal formation in central neural circuits

Current Techniques for Investigating the Brain Extracellular Space

The brain extracellular space (ECS) is a continuous reticular compartment that lies between the cells of the brain. It is vast in extent relative to its resident cells, yet, at the same time the nano- to micrometer dimensions of its channels and reservoirs are commonly finer than the smallest cellular structures. Our conventional view of this compartment as largely static and of secondary importance for brain function is rapidly changing, and its active dynamic roles in signaling and metabolite clearance have come to the fore. It is further emerging that ECS microarchitecture is highly heterogeneous and dynamic and that ECS geometry and diffusional properties directly modulate local diffusional transport, down to the nanoscale around individual synapses. The ECS can therefore be considered an extremely complex and diverse compartment, where numerous physiological events are unfolding in parallel on spatial and temporal scales that span orders of magnitude, from milliseconds to hours, and from nanometers to centimeters. To further understand the physiological roles of the ECS and identify new ones, researchers can choose from a wide array of experimental techniques, which differ greatly in their applicability to a given sample and the type of data they produce. Here, we aim to provide a basic introduction to the available experimental techniques that have been applied to address the brain ECS, highlighting their main characteristics. We include current gold-standard techniques, as well as emerging cutting-edge modalities based on recent super-resolution microscopy. It is clear that each technique comes with unique strengths and limitations and that no single experimental method can unravel the unknown physiological roles of the brain ECS on its own.This work was supported by the grants from the Spanish Ministry for Research and Innovation SAF2017-83776-R and RYC-2014-15994 to JT, IJCI-2017-32114 to FS, University of the Basque Country grant GIU18/094 to OP and JT, and a Basque Government grant PIBA 2019-65 to JT

QUANTITATIVE MODELING OF SCALING OF PATTERNS AND RECEPTOR SIGNALING IN MORPHOGENESIS

Organs and tissue development often experience perturbations, but developmental processes seem to replicate a common body template to maintain appropriate proportions and positions. The key signaling factors that guide a number of those processes are known as morphogens. Developing cells sense their respective positional information from a graded morphogen profile, and differentiate into patterns. Remarkably, patterns are highly robust and reproducible among species, and the underlying mechanisms associated with such high degrees of precision are still enigmatic. In addition, details of the signal, such as the Bone Morphogenetic Protein (BMP) signal, that transmit patterning information to a group of homogenous cells to differentiate is not well understood. Determining how developmental processes ensure robust patterning in the presence of perturbations, and what regulatory mechanisms act to ensure robust and reproducible patterning are two longstanding questions that need unraveling. Moreover, determining the mechanisms by which BMP heterodimers dominate signaling in developing zebrafish embryos and other contexts is a key factor in understanding developmental regulation for a classic morphogen patterning system. To answer these questions, this work has developed a set of mathematical models to evaluate and interrogate potential signaling networks and regulatory motifs. These models identify scaling mechanisms, test hypotheses on heterodimer dominance during signal transduction, and show how patterning systems function. For the scaling problem, this research proposes a Two Component System (TCS) mechanism, where a morphogen (m) and a modulator (M) interact to alter the transport and reaction properties of each other spatially. An exhaustive parametric and network motif screen is conducted for several TCS variants under the reaction-diffusion-advection paradigm with spatially varying coefficients. Our analysis revealed a number of candidate networks and minimal regulatory motifs that achieve the precision needed for a developing species to ensure perfect development. Computational models of patterning signal, namely the Bone Morphogenetic Protein (BMP) mediated signal, were developed to analyze the receptor oligomerization that forms heterotetrameric receptor associations in BMP signaling. The oligomerization model disproves previous kinetic based hypotheses of heterodimer dominance, and identifies other theoretical conditions to acquire heterodimer dominance. Finally, the model predicts that heterodimer dominance provides a larger dynamic range and a higher concentration of morphogen activity, making it a robust sensor responding wide ranges of morphogen concentrations fundamental to a morphogen gradient system. Moreover, stochastic analysis of oligomerization steps reveal that recruitment of type II receptors during the receptor oligomerization by itself does not tend to lower noise in receptor signaling. This outcome can be applied to develop a complete probabilistic model of receptor oligomerization events. The computational arrangements and frameworks developed in this research have wider applications - for instance, illustration of a large-scale screen of a reaction-diffusion-advection systems with spatially varying coefficients is an efficient strategy to perform a large-scale screen of such system and could have wider applications in other areas. Additionally, our mathematical framework on the dynamics of a tetrameric complex formation and oligomerization steps could be applicable to other signaling pathways that require trimeric/tetrameric complex formation on the cell surface to elicit signaling

DYNAMIC INTERACTIONS OF NODAL FACTORS AND THEIR RECEPTORS IN ZEBRAFISH EMBRYOS

Ph.DDOCTOR OF PHILOSOPH

The Effects of NR2 Subunit-Dependent NMDA Receptor Kinetics on Synaptic Transmission and CaMKII Activation

N-Methyl-d-aspartic acid (NMDA) receptors are widely expressed in the brain and are critical for many forms of synaptic plasticity. Subtypes of the NMDA receptor NR2 subunit are differentially expressed during development; in the forebrain, the NR2B receptor is dominant early in development, and later both NR2A and NR2B are expressed. In heterologous expression systems, NR2A-containing receptors open more reliably and show much faster opening and closing kinetics than do NR2B-containing receptors. However, conflicting data, showing similar open probabilities, exist for receptors expressed in neurons. Similarly, studies of synaptic plasticity have produced divergent results, with some showing that only NR2A-containing receptors can drive long-term potentiation and others showing that either subtype is capable of driving potentiation. In order to address these conflicting results as well as open questions about the number and location of functional receptors in the synapse, we constructed a Monte Carlo model of glutamate release, diffusion, and binding to NMDA receptors and of receptor opening and closing as well as a model of the activation of calcium-calmodulin kinase II, an enzyme critical for induction of synaptic plasticity, by NMDA receptor-mediated calcium influx. Our results suggest that the conflicting data concerning receptor open probabilities can be resolved, with NR2A- and NR2B-containing receptors having very different opening probabilities. They also support the conclusion that receptors containing either subtype can drive long-term potentiation. We also are able to estimate the number of functional receptors at a synapse from experimental data. Finally, in our models, the opening of NR2B-containing receptors is highly dependent on the location of the receptor relative to the site of glutamate release whereas the opening of NR2A-containing receptors is not. These results help to clarify the previous findings and suggest future experiments to address open questions concerning NMDA receptor function

Mechanistic and Statistical Models to Understand CXCL12/CXCR4/CXCR7 in Breast Cancer.

Signaling via the CXCL12/CXCR4 axis is instrumental to the metastasis of more than 20 cancers, yet blocking the pathway alone has not been effective as cancer therapy. Since cancer progression results from a complex network of interdependent biological events, preventing metastasis cannot be understood by studying only one gene or protein at a time. In this thesis, we employed mathematical and statistical models to examine complexity in the CXCL12/CXCR4/CXCR7 signaling axis. First, we performed a comprehensive analysis of CXCL12 isoform expression in breast cancer. This is the first study to correlate the expression levels of all six CXCL12 isoforms to cancer survival outcomes. Second, to understand mechanisms of physiological gradient formation, we built a hybrid agent-based model of cancer cell chemotaxis that links molecular scale events to chemokine gradient shaping and sensing. Third, to understand how co-expression of CXCR7 may alter CXCR4 signaling, we constructed a mechanistic model of CXCR4/CXCR7 receptor dynamics and signaling with an emphasis on shared signaling components. Themes arising from this work include the importance of non-specific binding of ligand to surfaces, receptor desensitization, gradient sensing, and compensatory effects resulting from the competition of shared signaling components.PhDChemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/111458/1/seiwon_1.pd