Quantifying How Staining Methods Bias Measurements of Neuron Morphologies

The process through which neurons are labeled is a key methodological choice in measuring neuron morphology. However, little is known about how this choice may bias measurements. To quantify this bias we compare the extracted morphology of neurons collected from the same rodent species, experimental condition, gender distribution, age distribution, brain region and putative cell type, but obtained with 19 distinct staining methods. We found strong biases on measured features of morphology. These were largest in features related to the coverage of the dendritic tree (e.g., the total dendritic tree length). Understanding measurement biases is crucial for interpreting morphological data

Distribution and regulation of ion channels in neurons: Quantitative studies of global ion channel transport and homeostatic synaptic scaling

A healthy neuron must continually produce millions of proteins and distribute them to function-specific regions of the cell. Among these proteins are ion channels that modulate neuronal excitability, allowing neurons to fulfill their primary role of information transfer. Neurons are unique among cells in their morphology, with projections that extend hundreds to thousands of microns. Neuron size and asymmetry pose a challenge for autoregulation of properties that require cargo transport across the cell. Homeostasis of ion channel localization has strong implications for neural excitability. This thesis concerns the intracellular distribution of ion channels in the context of longitudinal transport and global neuron regulation. The principal contributions are experimental measurements, data analysis, and modeling in the study of longitudinal neurite transport. Empirical investigations focus on the distribution and trafficking kinetics of ion channel Kv4.2, including quantitative measurements of both passive diffusion and active microtubule-based transport in both axons and dendrites (Chapters 3 and 5). Mass action models reveal that measured transport profiles corroborate discrepancies in Kv4.2 localization both between neurite types and along the somatodendritic axis (Chapter 4). Exchange between mobile and immobile fractions, inferred from analysis of repeated photobleaching, shapes intracellular distribution of Kv4.2 (Chapter 5). Further, the ensuing theoretical study surveys global regulation of ion channels, specifically for synaptic scaling, which requires cell-wide modulation of AMPA receptors for normalization of neural excitability. A unified model of synaptic potentiation, transport, and feedback reveals limitations imposed on synaptic scaling by neuron morphology. A neuron balances the stability, accuracy, and efficiency of synaptic scaling (Chapter 6).National Institutes of Health Oxford-Cambridge Scholars Gates Cambridge University of North Carolina Medical Scientist Training Progra

The cellular and synaptic architecture of the mechanosensory dorsal horn

The deep dorsal horn is a poorly characterized spinal cord region implicated in processing low-threshold mechanoreceptor (LTMR) information. We report an array of mouse genetic tools for defining neuronal components and functions of the dorsal horn LTMR-recipient zone (LTMR-RZ), a role for LTMR-RZ processing in tactile perception, and the basic logic of LTMR-RZ organization. We found an unexpectedly high degree of neuronal diversity in the LTMR-RZ: seven excitatory and four inhibitory subtypes of interneurons exhibiting unique morphological, physiological, and synaptic properties. Remarkably, LTMRs form synapses on between four and 11 LTMR-RZ interneuron subtypes, while each LTMR-RZ interneuron subtype samples inputs from at least one to three LTMR classes, as well as spinal cord interneurons and corticospinal neurons. Thus, the LTMR-RZ is a somatosensory processing region endowed with a neuronal complexity that rivals the retina and functions to pattern the activity of ascending touch pathways that underlie tactile perception

Unveiling the Neuromorphological Space

This article proposes the concept of neuromorphological space as the multidimensional space defined by a set of measurements of the morphology of a representative set of almost 6000 biological neurons available from the NeuroMorpho database. For the first time, we analyze such a large database in order to find the general distribution of the geometrical features. We resort to McGhee's biological shape space concept in order to formalize our analysis, allowing for comparison between the geometrically possible tree-like shapes, obtained by using a simple reference model, and real neuronal shapes. Two optimal types of projections, namely, principal component analysis and canonical analysis, are used in order to visualize the originally 20-D neuron distribution into 2-D morphological spaces. These projections allow the most important features to be identified. A data density analysis is also performed in the original 20-D feature space in order to corroborate the clustering structure. Several interesting results are reported, including the fact that real neurons occupy only a small region within the geometrically possible space and that two principal variables are enough to account for about half of the overall data variability. Most of the measurements have been found to be important in representing the morphological variability of the real neurons

GliaMorph: A modular image analysis toolkit to quantify Müller glial cell morphology

Cell morphology is critical for all cell functions. This is particularly true for glial cells as they rely on complex shape to contact and support neurons. However, methods to quantify complex glial cell shape accurately and reproducibly are lacking. To address this, we developed the image analysis pipeline "GliaMorph". GliaMorph is a modular analysis toolkit developed to perform (i) image pre-processing, (ii) semi-automatic region-of-interest (ROI) selection, (iii) apicobasal texture analysis, (iv) glia segmentation, and (v) cell feature quantification. Müller Glia (MG) have a stereotypic shape linked to their maturation and physiological status. We here characterized MG on three levels, including (a) global image-level, (b) apicobasal texture, and (c) regional apicobasal vertical-to-horizontal alignment. Using GliaMorph we quantified MG development on a global and single-cell level, showing increased feature elaboration and subcellular morphological rearrangement in the zebrafish retina. As proof-of-principle, we analysed expression changes in a mouse glaucoma model, identifying subcellular protein localization changes in MG. Together, GliaMorph enables an in-depth understanding of MG morphology in the developing and diseased retina

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

An analysis of taste neuron morphological variability: influences of neuron type and plasticity.

Taste neurons are functionally and molecularly diverse, but their morphological diversity was, until recently, completely unexplored. Taste neurons were considered relay cells, communicating information from taste-transducing cells to the brain without variation in morphology. Instead, individual taste neurons are tremendously morphologically variable. To determine how differences in branching relate to the number and types of taste-transducing cells providing neuronal input, I combined sparse cell genetic labeling with a whole-mount immunohistochemistry and analysis workflow. I found that the maximum number of taste-transducing cells capable of providing convergent input onto individual gustatory neurons varied with a range of 1-22 taste-transducing cells. Consistently, simple taste neurons contact a few taste-transducing cells of the same type (either sour or sweet/bitter/umami), whereas branched neurons contact many taste-transducing cells of both types. These results suggest differential convergence within the peripheral taste system in the type(s) of taste-transducing cells providing input and the number of taste-transducing cells of the same type. Taste arbors (the portion of the neuron within the taste bud) also vary in morphology and the number and types of taste-transducing cells contacted. I reconstructed 151 taste arbors from the full taste neuron population to analyze differences in size, complexity, symmetry and number and type to taste-transducing cells contacted. I determined that these features are not determined by the size or cellular composition of the taste bud. Taste arbors exist on a continuum of complexity, not in discrete categories of stereotyped endings. Large, asymmetrical arbors contacted more taste-transducing cells of both types, whereas smaller, symmetrical arbors contacted 1-2 taste-transducing cells. Differences in arbor complexity are consistent with regulation by plasticity rather than neuron type. To test this idea, I examined the arbors and ganglion neurons of the first molecularly and functionally described taste neuron type defined by the expression preproenkephalin (Penk). The arbors of Penk neurons do not represent a discrete subset of the arbor morphologies present in the full population. However, more symmetrical arbors from Penk+ neurons contact two taste-transducing cells of different types than the full population. All Penk+ ganglion neurons contact cells of both types while consistently contacting more Car4+ cells than PLCβ2+ cells. Together, these results indicate that plasticity dictates arbor morphology, whereas neuron type influences the number and type of taste-transducing cells providing input

Regenerative capacity in the lamprey spinal cord is not altered after a repeated transection

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS One 14(1), (2019):e0204193, doi: 10.1371/journal.pone.0204193.The resilience of regeneration in vertebrates is not very well understood. Yet understanding if tissues can regenerate after repeated insults, and identifying limitations, is important for elucidating the underlying mechanisms of tissue plasticity. This is particularly challenging in tissues, such as the nervous system, which possess a large number of terminally differentiated cells and often exhibit limited regeneration in the first place. However, unlike mammals, which exhibit very limited regeneration of spinal cord tissues, many non-mammalian vertebrates, including lampreys, bony fishes, amphibians, and reptiles, regenerate their spinal cords and functionally recover even after a complete spinal cord transection. It is well established that lampreys undergo full functional recovery of swimming behaviors after a single spinal cord transection, which is accompanied by tissue repair at the lesion site, as well as axon and synapse regeneration. Here we begin to explore the resilience of spinal cord regeneration in lampreys after a second spinal transection (re-transection). We report that by all functional and anatomical measures tested, lampreys regenerate after spinal re-transection just as robustly as after single transections. Recovery of swimming, synapse and cytoskeletal distributions, axon regeneration, and neuronal survival were nearly identical after spinal transection or re-transection. Only minor differences in tissue repair at the lesion site were observed in re-transected spinal cords. Thus, regenerative potential in the lamprey spinal cord is largely unaffected by spinal re-transection, indicating a greater persistent regenerative potential than exists in some other highly regenerative models. These findings establish a new path for uncovering pro-regenerative targets that could be deployed in non-regenerative conditions.The authors would like to thank Dr. Cristina Roman-Vendrell and Louie Kerr, Director of the Central Microscopy Facility at the MBL, for technical support. We also thank Dr. Juan Diaz-Quiroz for helpful comments on the manuscript. EG was supported in part by an NSF REU Award (#1659604: Biological Discovery in Woods Hole at the Marine Biological Laboratory)