Image informatics strategies for deciphering neuronal network connectivity

Brain function relies on an intricate network of highly dynamic neuronal connections that rewires dramatically under the impulse of various external cues and pathological conditions. Among the neuronal structures that show morphologi- cal plasticity are neurites, synapses, dendritic spines and even nuclei. This structural remodelling is directly connected with functional changes such as intercellular com- munication and the associated calcium-bursting behaviour. In vitro cultured neu- ronal networks are valuable models for studying these morpho-functional changes. Owing to the automation and standardisation of both image acquisition and image analysis, it has become possible to extract statistically relevant readout from such networks. Here, we focus on the current state-of-the-art in image informatics that enables quantitative microscopic interrogation of neuronal networks. We describe the major correlates of neuronal connectivity and present workflows for analysing them. Finally, we provide an outlook on the challenges that remain to be addressed, and discuss how imaging algorithms can be extended beyond in vitro imaging studies

Super-Resolution STED Microscopy in live Brain Tissue

STED microscopy is one of several fluorescence microscopy techniques that permit imaging at higher spatial resolution than what the diffraction-limit of light dictates. STED imaging is unique among these super-resolution modalities in being a beam-scanning microscopy technique based on confocal or 2-photon imaging, which provides the advantage of superior optical sectioning in thick samples. Compared to the other super-resolution techniques that are based on widefield microscopy, this makes STED particularly suited for imaging inside live brain tissue, such as in slices or in vivo. Notably, the 50nm resolution provided by STED microscopy enables analysis of neural morphologies that conventional confocal and 2-photon microscopy approaches cannot resolve, including all-important synaptic structures. Over the course of the last 20years, STED microscopy has undergone extensive developments towards ever more versatile use, and has facilitated remarkable neurophysiological discoveries. The technique is still not widely adopted for live tissue imaging, even though one of its particular strengths is exactly in resolving the nanoscale dynamics of synaptic structures in brain tissue, as well as in addressing the complex morphologies of glial cells, and revealing the intricate structure of the brain extracellular space. Not least, live tissue STED microscopy has so far hardly been applied in settings of pathophysiology, though also here it shows great promise for providing new insights. This review outlines the technical advantages of STED microscopy for imaging in live brain tissue, and highlights key neurobiological findings brought about by the technique.The authors acknowledge funding for their general work from the Spanish Ministry of Science and Innovation (SAF-2017-83776-R, RYC-2014-15994 and IJCI-2017-32114), the Basque Government (PIBA19-0065 and PIBA-2020-1-0061), and the University of the Basque Country (GIU18/094 and INF19-29

Technologies for imaging neural activity in large volumes

Neural circuitry has evolved to form distributed networks that act dynamically across large volumes. Collecting data from individual planes, conventional microscopy cannot sample circuitry across large volumes at the temporal resolution relevant to neural circuit function and behaviors. Here, we review emerging technologies for rapid volume imaging of neural circuitry. We focus on two critical challenges: the inertia of optical systems, which limits image speed, and aberrations, which restrict the image volume. Optical sampling time must be long enough to ensure high-fidelity measurements, but optimized sampling strategies and point spread function engineering can facilitate rapid volume imaging of neural activity within this constraint. We also discuss new computational strategies for the processing and analysis of volume imaging data of increasing size and complexity. Together, optical and computational advances are providing a broader view of neural circuit dynamics, and help elucidate how brain regions work in concert to support behavior

Développement d'un microscope STED à excitation deux photons et son application aux neurosciences

The advent of STED microscopy has created a lot of excitement in the field of neuroscience becausemany important neuronal structures, such as dendritic spines, axonal shafts or astroglial processes,cannot be properly resolved by regular light microscopy techniques. Two-photon fluorescence microscopy is a widely used imaging technique in neuroscience because it permits imaging dynamic events deep inside light-scattering brain tissue, providing high optical sectioning and depth penetration. However, the spatial resolution of this approach is limited to around half a micron, and hence is inadequate for revealing many morphological details of neurons and synapses. The aim of my PhD work was to A) develop a microscope that improves on two-photon imaging by combining it with STED microscopy and to B) demonstrate its potential for nanoscale imaging of dynamic neural processes in acute brain slices and in vivo. The new microscope achieves a lateral spatial resolution of ~50 nm at imaging depths of ~50 μm in living brain slices. It works with green fluorophores, including common fluorescent proteins like GFP and YFP, offering two-color contrast based on spectral detection and linear unmixing. Because of its upright design using a long working distance water-immersion objective, it was possible to incorporate electrophysiological techniques like patch-clamping or to add a stage for in vivo imaging. I have used the new microscope to image fine neural processes and their nanoscale dynamics in different experimental preparations and brain regions, revealing new and interesting morphological features of dendrites and spines. In addition, I have explored different labeling strategies to be able to use STED microscopy for visualizing protein trafficking and dynamics at the nanoscale in brain slices.L’avènement de la microscopie STED (Stimulated Emission Depletion) a bouleversé le domaine desneurosciences du au fait que beaucoup de structures neuronale, tels que les épines dendritiques, lesaxones ou les processus astrocytaires, ne peuvent pas être correctement résolu en microscopiephotonique classique. La microscopie 2-photon est une technique d’imagerie photonique très largement utilisée dans le domaine des neurosciences car elle permet d’imager les événements dynamique en profondeur dans le tissu cérébral, offrant un excellent sectionnement optique et une meilleure profondeur de pénétration. Cependant, la résolution spatiale de cette approche est limitée autour de 0.5 μm, la rendant inappropriée pour étudier les détails morphologiques des neurones et synapses. Le but de mon travail de thèse était à A) développer un microscope qui permet d'améliorer l'imagerie 2-photon en la combinant avec la microscopie STED et B) démontrer son potentiel pour l'imagerie à l'échelle nanométrique de processus neuronaux dynamiques dans des tranches de cerveau aigus et in vivo. Le nouveau microscope permet d'obtenir une résolution spatiale latérale de ~ 50 nm à des profondeurs d'imagerie de ~ 50 μm dans du tissu cérébral vivant. Il fonctionne avec des fluorophores verts, y compris les protéines fluorescentes communes telles que la GFP et YFP, offrant le contraste de deux couleurs basé sur la détection spectrale et linéaire ‘unmixing’. S’agissant d’un microscope droit, utilisant un objectif à immersion ayant une grande distance de travail, nous avons pu incorporer des techniques électrophysiologiques comme patch-clamp et ajouter une plateforme pour l'imagerie in vivo. J’ai utilise ce nouveau microscope pour imager des processus neuronaux fins et leur dynamique à l’échelle nanométrique dans différent types de préparations et des régions différentes du cerveau. J’ai pu révéler des nouvelles caractéristiques morphologique des dendrites et épines. En outre, j'ai exploré différentes stratégies de marquage pour pouvoir utiliser la microscopie STED pour imager le trafic des protéines et de leur dynamique à l'échelle nanométrique dans des tranches de cerveau

Improved methods for functional neuronal imaging with genetically encoded voltage indicators

Voltage imaging has the potential to revolutionise neuronal physiology, enabling high temporal and spatial resolution monitoring of sub- and supra-threshold activity in genetically defined cell classes. Before this goal is reached a number of challenges must be overcome: novel optical, genetic, and experimental techniques must be combined to deal with voltage imaging’s unique difficulties. In this thesis three techniques are applied to genetically encoded voltage indicator (GEVI) imaging. First, I describe a multifocal two-photon microscope and present a novel source localisation control and reconstruction algorithm to increase scattering resistance in functional imaging. I apply this microscope to image population and single-cell voltage signals from voltage sensitive fluorescent proteins in the first demonstration of multifocal GEVI imaging. Second, I show that a recently described genetic technique that sparsely labels cortical pyramidal cells enables single-cell resolution imaging in a one-photon widefield imaging configuration. This genetic technique allows simple, high signal-to-noise optical access to the primary excitatory cells in the cerebral cortex. Third, I present the first application of lightfield microscopy to single cell resolution neuronal voltage imaging. This technique enables single-shot capture of dendritic arbours and resolves 3D localised somatic and dendritic voltage signals. These approaches are finally evaluated for their contribution to the improvement of voltage imaging for physiology.Open Acces

Large-scale circuit reconstruction in medial entorhinal cortex

Es ist noch weitgehend ungeklärt, mittels welcher Mechanismen die elektrische Aktivität von Nervenzellpopulationen des Gehirns Verhalten ermöglicht. Die Orientierung im Raum ist eine Fähigkeit des Gehirns, für die im Säugetier der mediale entorhinale Teil der Großhirnrinde als entscheidende Struktur identifiziert wurde. Hier wurden Nervenzellen gefunden, die die Umgebung des Individuums in einer gitterartigen Anordnung repräsentieren. Die neuronalen Schaltkreise, welche diese geordnete Nervenzellaktivität im medialen entorhinalen Kortex (MEK) ermöglichen, sind noch wenig verstanden. Die vorliegende Dissertation hat eine Klärung der zellulären Architektur und der neuronalen Schaltkreise in der zweiten Schicht des MEK der Ratte zum Ziel. Zunächst werden die Beiträge zur Entdeckung der hexagonal angeordneten zellulären Anhäufungen in Schicht 2 des MEK sowie zur Beschreibung der Dichotomie der Haupt-Nervenzelltypen dargestellt. Im zweiten Teil wird erstmalig eine konnektomische Analyse des MEK beschrieben. Die detaillierte Untersuchung der Architektur einzelner exzitatorischer Axone ergab das überraschende Ergebnis der präzisen Sortierung von Synapsen entlang axonaler Pfade. Die neuronalen Schaltkreise, in denen diese Neurone eingebettet sind, zeigten eine starke zeitliche Bevorzugung der hemmenden Neurone. Die hier erhobenen Daten tragen zu einem detaillierteren Verständnis der neuronalen Schaltkreise im MEK bei. Sie enthalten die erste Beschreibung überraschend präziser axonaler synaptischer Ordnung im zerebralen Kortex der Säugetiere. Diese Schaltkreisarchitektur lässt einen Effekt auf die Weiterleitung synchroner elektrischer Populationsaktivität im MEK vermuten. In zukünftigen Studien muss insbesondere geklärt werden, ob es sich bei den hier berichteten Ergebnissen um eine Besonderheit des MEK oder ein generelles Verschaltungsprinzip der Hirnrinde des Säugetiers handelt.The mechanisms by which the electrical activity of ensembles of neurons in the brain give rise to an individual’s behavior are still largely unknown. Navigation in space is one important capacity of the brain, for which the medial entorhinal cortex (MEC) is a pivotal structure in mammals. At the cellular level, neurons that represent the surrounding space in a grid-like fashion have been identified in MEC. These so-called grid cells are located predominantly in layer 2 (L2) of MEC. The detailed neuronal circuits underlying this unique activity pattern are still poorly understood. This thesis comprises studies contributing to a mechanistic description of the synaptic architecture in rat MEC L2. First, this thesis describes the discovery of hexagonally arranged cell clusters and anatomical data on the dichotomy of the two principle cell types in L2 of the MEC. Then, the first connectomic study of the MEC is reported. An analysis of the axonal architecture of excitatory neurons revealed synaptic positional sorting along axons, integrated into precise microcircuits. These microcircuits were found to involve interneurons with a surprising degree of axonal specialization for effective and fast inhibition. Together, these results contribute to a detailed understanding of the circuitry in MEC. They provide the first description of highly precise synaptic arrangements along axons in the cerebral cortex of mammals. The functional implications of these anatomical features were explored using numerical simulations, suggesting effects on the propagation of synchronous activity in L2 of the MEC. These findings motivate future investigations to clarify the contribution of precise synaptic architecture to computations underlying spatial navigation. Further studies are required to understand whether the reported synaptic specializations are specific for the MEC or represent a general wiring principle in the mammalian cortex

Optical imaging of dendritic spikes in apical oblique dendrites of layer 5 pyramidal neurons

Dendrites have active properties capable of generating dendritic spikes that could boost the impact of distal synaptic inputs. The strong passive filtering of the membrane and generation of local dendritic spikes enable different dendritic regions to function as independent computational compartments. While there is a wealth of information about cortical processing in apical tuft and basal dendrites, the functional role of apical oblique dendrites of layer 5 pyramidal neurons (L5PNs) are less understood. In this thesis, I aim to understand to functional role of thin apical oblique dendrites of L5PNs in the cortex. Using a previously published multi-compartment model of a L5PN, I first investigated the excitability of apical oblique dendrites and the extent of action potential (AP) back-propagation. In the model, I found that a 2-AP train at f > 35 Hz elicited an oblique branch spike in certain dendrites. The spike is mediated by activation of voltage-gated sodium and voltage-gated calcium channels. In addition, oblique branch spikes manifest as after-depolarizing potentials (ADP) at the soma. I then experimentally verified the generation of spikes by imaging the dendritic activity of oblique branches of L5PNs from the somatosensory cortex in vitro. I used our custom-built two-photon (2P) holographic microscope to perform functional calcium imaging on thin oblique branches of L5PNs in vitro. Oblique branch spikes are evoked at a critical frequency of fc = 57+/-5 Hz (from calcium imaging) and fc = 72+/-4 Hz (from ADP measurements) of a 2-AP train. Generation of spikes in oblique dendrites could establish their role as independent computational compartments that could boost coincident synaptic inputs. To further improve optical recording along dendrites, I proposed novel optical recording techniques to enhance the signal-to-noise ratio (SNRs) of the detected signals. A 4-fold improvement in the SNR was obtained with temporal gating for multi-site holographic calcium imaging, while a 6-fold increase in SNR was obtained for voltage imaging when using scattered photons to excite voltage indicators. These two novel SNR enhancement techniques can facilitate experiments that require imaging of activity in thin dendrites