3D Reconstruction and Standardization of the Rat Vibrissal Cortex for Precise Registration of Single Neuron Morphology

Author Summary For studying the neural basis of perception and behavior, it would be ideal to directly monitor sensory-evoked excitation streams within neural circuits, at sub-cellular and millisecond resolution. To do so, reverse engineering approaches of reconstructing circuit anatomy and synaptic wiring have been suggested. The resulting anatomically realistic models may then allow for computer simulations (in silico experiments) of circuit function. A natural starting point for reconstructing neural circuits is a cortical column, which is thought to be an elementary functional unit of sensory cortices. In the vibrissal area of rodent somatosensory cortex, a cytoarchitectonic equivalent, designated as a ‘barrel column’, has been described. By reconstructing the 3D geometry of almost 1,000 barrel columns, we show that the somatotopic layout of the vibrissal cortex is highly preserved across animals. This allows generating a standard cortex and registering neuron morphologies, obtained from different experiments, to their ‘true’ location. Marking a crucial step towards reverse engineering of cortical circuits, the present study will allow estimating synaptic connectivity within an entire cortical area by structural overlap of registered axons and dendrites

From single cells and single columns to cortical networks: dendritic excitability, coincidence detection and synaptic transmission in brain slices and brains

Although patch pipettes were initially designed to record extracellularly the elementary current events from muscle and neuron membranes, the whole-cell and loose cell-attached recording configurations proved to be useful tools for examination of signalling within and between nerve cells. In this Paton Prize Lecture, I will initially summarize work on electrical signalling within single neurons, describing communication between the dendritic compartments, soma and nerve terminals via forward- and backward-propagating action potentials. The newly discovered dendritic excitability endows neurons with the capacity for coincidence detection of spatially separated subthreshold inputs. When these are occurring during a time window of tens of milliseconds, this information is broadcast to other cells by the initiation of bursts of action potentials (AP bursts). The occurrence of AP bursts critically impacts signalling between neurons that are controlled by target-cell-specific transmitter release mechanisms at downstream synapses even in different terminals of the same neuron. This can, in turn, induce mechanisms that underly synaptic plasticity when AP bursts occur within a short time window, both presynaptically in terminals and postsynaptically in dendrites. A fundamental question that arises from these findings is: what are the possible functions of active dendritic excitability with respect to network dynamics in the intact cortex of behaving animals?' To answer this question, I highlight in this review the functional and anatomical architectures of an average cortical column in the vibrissal (whisker) field of the somatosensory cortex (vS1), with an emphasis on the functions of layer 5 thick-tufted cells (L5tt) embedded in this structure. Sensory-evoked synaptic and action potential responses of these major cortical output neurons are compared with responses in the afferent pathway, viz. the neurons in primary somatosensory thalamus and in one of their efferent targets, the secondary somatosensory thalamus. Coincidence-detection mechanisms appear to be implemented in vivo as judged from the occurrence of AP bursts. Three-dimensional reconstructions of anatomical projections suggest that inputs of several combinations of thalamocortical projections and intra- and transcolumnar connections, specifically those from infragranular layers, could trigger active dendritic mechanisms that generate AP bursts. Finally, recordings from target cells of a column reveal the importance of AP bursts for signal transfer to these cells. The observations lead to the hypothesis that in vS1 cortex, the sensory afferent sensory code is transformed, at least in part, from a rate to an interval (burst) code that broadcasts the occurrence of whisker touch to different targets of L5tt cells. In addition, the occurrence of pre- and postsynaptic AP bursts may, in the long run, alter touch representation in cortex

Simulation of sensory-evoked signal flow in anatomically realistic models of neural networks

In this thesis, a new concept for development and simulation of anatomically and functionally constrained models of signal flow in neural networks is described. This approach consists of the following tools: 1. A standardized anatomical reference frame of the brain region studied and registration methods to integrate anatomical data from different experiments with the highest precision possible. 2. A method for determining morphological neuron types to allow correlation between measurements of the morphology and functional responses of individual neurons. 3. A tool to build an average three-dimensional (3D) statistical model of the neural networks in a brain region based on a representative sparse sample of all neuron types present in the brain region. This model contains 3D morphological models for every neuron in the brain region, as well as the total number and 3D distribution of synaptic contacts between them. 4. A method to activate the network based on measured responses of different neuron types, and to simulate the response of individual neurons representative of different cell types within this network model. The feasibility and validity of this process is demonstrated on the example of rat vibrissal cortex. The 3D model of this primary sensory area in cortex contains ∼ 530,000 neurons of 16 different types and ∼ 6 × 10^9 thalamocortical and intracortical synapses. Activation of this model with functional responses measured after whisker touch and simulation of the responses of different neuron types shows that the simulated model responses match experimental measurements. This allowed investigating how robust sensory-evoked responses after different sensory stimuli are formed in different neuron types using computer simulations, and to make predictions to experimentally test these hypotheses.Diese Dissertation beschreibt einen neuartigen Ansatz zur Entwicklung und Simulation von Modellen des Signalflusses in neuronalen Netzwerken unter anatomisch und funktionell realistischen Randbedingungen. Dieser Ansatz besteht aus den folgenden Methoden: 1. Ein standardisiertes anatomisches Referenzsystem der betrachteten Hirnregion und Registrierungsmethoden die es erlauben anatomische Daten aus unterschiedlichen Experimenten mit höchstmöglicher Genauigkeit zu integrieren. 2. Eine Methode zur Bestimmung morphologischer Typen von Nervenzellen um Messungen von der Morphologie und funktioneller Antworten einzelner Nervenzellen in Bezug zu setzen. 3. Eine Methode um ein mittleres dreidimensionales (3D) statistisches Modell der neuronalen Netzwerke in einer Hirnregion zu bauen, das auf einer repräsentativen Stichprobe aller Nervenzelltypen in dieser Hirnregion beruht. Dieses Modell beinhaltet 3D morphologische Modelle für jede Nervenzelle in der Hirnregion, und die Zahl und 3D Verteilung synaptischer Verknüpfungen zwischen diesen. 4. Eine Methode um dieses Netzwerk aufgrund von gemessenen Antworten unterschiedlicher Nervenzelltypen zu aktivieren, und die Antwort einzelner repräsentativer Nervenzellen bestimmten Typs innerhalb dieses Netzwerkmodells zu simulieren. Die Machbarkeit und Gültigkeit dieses Ansatzes wird am Beispiel des Tasthaarsystems im Kortex der Ratte demonstriert. Das 3D Modell dieses primären sensorischen Kortex enthält ∼ 530000 Nervenzellen von 16 unterschiedlichen Typen und ∼ 6 × 10^9 thalamokortikale und intrakortikale Synapsen. Aktivierung dieses Modells mit gemessen funktionellen Antworten auf passive Berührung eines Schnurrhaares und Simulation der Antworten unterschiedlicher Nervenzelltypen zeigt dass die simulierten Antworten mit experimentellen Messungen übereinstimmen. Dies erlaubt es mit Hilfe von Computersimulationen zu untersuchen wie robuste Antworten auf unterschiedliche Sinnesreize in unterschiedlichen Nervenzelltypen entstehen, und experimentell überprüfbare Vorhersagen zu machen

Dynamics of population activity in rat sensory cortex: Network correlations predict anatomical arrangement and information content

To study the spatiotemporal dynamics of neural activity in a cortical population, we implanted a 10 × 10 microelectrode array in the vibrissal cortex of urethane-anesthetized rats. We recorded spontaneous neuronal activity as well as activity evoked in response to sustained and brief sensory stimulation. To quantify the temporal dynamics of activity, we computed the probability distribution function (PDF) of spiking on one electrode given the observation of a spike on another. The spike-triggered PDFs quantified the strength, temporal delay, and temporal precision of correlated activity across electrodes. Nearby cells showed higher levels of correlation at short delays, whereas distant cells showed lower levels of correlation, which tended to occur at longer delays. We found that functional space built based on the strength of pairwise correlations predicted the anatomical arrangement of electrodes. Moreover, the correlation profile of electrode pairs during spontaneous activity predicted the “signal” and “noise” correlations during sensory stimulation. Finally, mutual information analyses revealed that neurons with stronger correlations to the network during spontaneous activity, conveyed higher information about the sensory stimuli in their evoked response. Given the 400-μm-distance between adjacent electrodes, our functional quantifications unravel the spatiotemporal dynamics of activity among nearby and distant cortical columns

The impact of neuron morphology on cortical network architecture

The neurons in the cerebral cortex are not randomly interconnected. This specificity in wiring can result from synapse formation mechanisms that connect neurons, depending on their electrical activity and genetically defined identity. Here, we report that the morphological properties of the neurons provide an additional prominent source by which wiring specificity emerges in cortical networks. This morphologically determined wiring specificity reflects similarities between the neurons’ axo-dendritic projections patterns, the packing density, and the cellular diversity of the neuropil. The higher these three factors are, the more recurrent is the topology of the network. Conversely, the lower these factors are, the more feedforward is the network’s topology. These principles predict the empirically observed occurrences of clusters of synapses, cell type-specific connectivity patterns, and nonrandom network motifs. Thus, we demonstrate that wiring specificity emerges in the cerebral cortex at subcellular, cellular, and network scales from the specific morphological properties of its neuronal constituents

Visualization of mouse barrel cortex using ex-vivo track density imaging

We describe the visualization of the barrel cortex of the primary somatosensory area (S1) of ex vivo adult mouse brain with short-tracks track density imaging (stTDI). stTDI produced much higher definition of barrel structures than conventional fractional anisotropy (FA), directionally-encoded color FA maps, spin-echo and T2-weighted imaging and gradient echo Ti/T2*-weighted imaging. 3D high angular resolution diffusion imaging (HARDI) data were acquired at 48 micron isotropic resolution for a (3 mm)3 block of cortex containing the barrel field and reconstructed using stTDI at 10 micron isotropic resolution. HARDI data were also acquired at 100 micron isotropic resolution to image the whole brain and reconstructed using stTDI at 20 micron isotropic resolution. The 10 micron resolution stTDI maps showed exceptionally clear delineation of barrel structures. Individual barrels could also be distinguished in the 20 micron stTDI maps but the septa separating the individual barrels appeared thicker compared to the 10 micron maps, indicating that the ability of stTDI to produce high quality structural delineation is dependent upon acquisition resolution. Close homology was observed between the barrel structure delineated using stTDI and reconstructed histological data from the same samples. stTDI also detects barrel deletions in the posterior medial barrel sub-field in mice with infraorbital nerve cuts. The results demonstrate that stTDI is a novel imaging technique that enables three-dimensional characterization of complex structures such as the barrels in S1 and provides an important complementary non-invasive imaging tool for studying synaptic connectivity, development and plasticity of the sensory system. (C) 2013 Elsevier Inc. All rights reserved

A proposal for a coordinated effort for the determination of brainwide neuroanatomical connectivity in model organisms at a mesoscopic scale

In this era of complete genomes, our knowledge of neuroanatomical circuitry remains surprisingly sparse. Such knowledge is however critical both for basic and clinical research into brain function. Here we advocate for a concerted effort to fill this gap, through systematic, experimental mapping of neural circuits at a mesoscopic scale of resolution suitable for comprehensive, brain-wide coverage, using injections of tracers or viral vectors. We detail the scientific and medical rationale and briefly review existing knowledge and experimental techniques. We define a set of desiderata, including brain-wide coverage; validated and extensible experimental techniques suitable for standardization and automation; centralized, open access data repository; compatibility with existing resources, and tractability with current informatics technology. We discuss a hypothetical but tractable plan for mouse, additional efforts for the macaque, and technique development for human. We estimate that the mouse connectivity project could be completed within five years with a comparatively modest budget.Comment: 41 page

Three-dimensional scanless holographic optogenetics with temporal focusing (3D-SHOT).

Optical methods capable of manipulating neural activity with cellular resolution and millisecond precision in three dimensions will accelerate the pace of neuroscience research. Existing approaches for targeting individual neurons, however, fall short of these requirements. Here we present a new multiphoton photo-excitation method, termed three-dimensional scanless holographic optogenetics with temporal focusing (3D-SHOT), which allows precise, simultaneous photo-activation of arbitrary sets of neurons anywhere within the addressable volume of a microscope. This technique uses point-cloud holography to place multiple copies of a temporally focused disc matching the dimensions of a neurons cell body. Experiments in cultured cells, brain slices, and in living mice demonstrate single-neuron spatial resolution even when optically targeting randomly distributed groups of neurons in 3D. This approach opens new avenues for mapping and manipulating neural circuits, allowing a real-time, cellular resolution interface to the brain

Segmentation and 3D reconstruction of animal tissues in histological images

Histology is considered the "gold standard" to access anatomical informationat a cellular level. In histological studies, tissue samples are cut into very thinsections, stained, and observed under a microscope by a specialist. Such studies,mainly concerning tissue structures, cellular components and their interactions, canbe useful to detect and diagnose certain pathologies. Thus, to find new techniquesand computational solutions to assist this diagnosis, such as the 3D image basedtissue reconstruction, is extremely interesting. In this chapter, a methodology tobuild 3D models from histological images is proposed, and the results obtained usingthis methodology in four experimental cases are presented and discussed based onquantitative and qualitative metrics