Reappraisal of transcallosal neuron organization in mice and evaluation of their dendritic remodeling and circuit integration following traumatic brain injury

Traumatic Brain Injury (TBI) is an enormous global socio-economic burden since, apart from its high death rate, it is the primary cause of coma worldwide and a prevalent cause of long-term disability. Until today there is no established treatment for dealing with the long-term outcomes of TBI despite many years of research. Although a lot is known about the pathophysiology of TBI in the damaged tissue and the surrounding area in case of focal lesion, only few studies have investigated the structural and functional integrity of the contralateral intact cortex. In order to explore this territory, this study employs a well-established and widely used animal model of focal open skull TBI known as the Controlled Cortical Impact (CCI) model. The first aim of this study was to systematically characterize a specific neuronal population, the transcallosal projection neurons, as they are the ones connecting the intact cortex with the lesioned cortex. The description of the organization of transcallosal neurons and their axonal projections at the contralateral hemisphere was carried out in healthy, non-injured C57Bl6 mice. Retrograde and anterograde tracing methods were implemented to label transcallosal cell bodies and their axonal projections, respectively. In addition, different injection coordinates were used in order to label transcallosal connections at distinct brain regions, including the motor cortex (M1), somatosensory cortex (S1), and barrel cortex, rostral and caudal to Bregma. In agreement with previous research, I observed that transcallosal projections are organized homotopically across the various brain regions, with the axonal terminals spanning the entire cortical column. Interestingly my study describes for the first time a non-negligible fraction of heterotopic transcallosal neurons that, in addition, display a slightly less strict layer distribution pattern compared to the homotopic ones. After the initial characterization of transcallosal neuron organization, I proceeded by investigating how these neurons with projections at the injury site are affected at various timepoints following focal TBI. I used GFPM mice to visualize dendrites and spines of transcallosal and non-transcallosal neurons, in order to examine their structural integrity at different timepoints post-injury. I detected significant differences in dendritic spine density and morphology between controls and injured mice, which were time-dependent. More specifically, the dendritic spine density in transcallosal neurons was strongly decreased as soon as 7days following injury. Interestingly, spine density in non-transcallosal neurons was not changed following TBI. In terms of spine shape, I found a morphological shift only for the apical tuft segments. These results point towards a general sensitivity of transcallosal spines to TBI-induced damage, where loss of spines (preferentially mature) seems to take place at 1-2 weeks post-injury and resolve at 3-6 weeks post-injury, indicative of late plasticity processes. As the anatomically connected neuronal population seems to recover overtime I then decided to further explore whether transcallosal circuit remodeling takes place after TBI. To do so I used the retrograde mono-trans-synaptic tracer SADΔG-GFP (EnvA) Rabies virus. In that way, I was able to distinctively label transcallosal neurons and their presynaptic partners and obtain an overview of the presynaptic population throughout the cortex across brain regions at different post-injury timepoints. This study demonstrates that spine plasticity did not result in adaptive circuit plasticity with the recruitment of other brain regions but rather that initial circuits were re-established. In brief, during this thesis I have demonstrated the adaptive plastic capacities of anatomically connected neurons to the brain injury. I believe that this knowledge may help in unraveling further compensatory plastic mechanisms that could then be therapeutically targeted to improve the outcome following brain injury

3D Architectural Analysis of Neurons, Astrocytes, Vasculature & Nuclei in the Motor and Somatosensory Murine Cortical Columns

Characterization of the complex cortical structure of the brain at a cellular level is a fundamental goal of neuroscience which can provide a better understanding of both normal function as well as disease state progression. Many challenges exist however when carrying out this form of analysis. Immunofluorescent staining is a key technique for revealing 3-dimensional structure, but subsequent fluorescence microscopy is limited by the quantity of simultaneous targets that can be labeled and intrinsic lateral and isotropic axial point-spread function (PSF) blurring during the imaging process in a spectral and depth-dependent manner. Even after successful staining, imaging and optical deconvolution, the sheer density of filamentous processes in the neuropil significantly complicates analysis due to the difficulty of separating individual cells in a highly interconnected network of tightly woven cellular arbors. In order to solve these problems, a variety of methodologies were developed and validated for improved analysis of cortical anatomy. An enhanced immunofluorescent staining and imaging protocol was utilized to precisely locate specific functional regions within brain slices at high magnification and collect four-channel, complete cortical columns. A powerful deconvolution routine was established which collected depth variant PSFs using an optical phantom for image restoration. Fractional volume analysis (FVA) was used to provide preliminary data of the proportions of each stained component in order to statistically characterize the variability within and between the functional regions in a depth-dependent and depth-independent manner. Finally, using machine learning techniques, a supervised learning model was developed that could automatically classify neuronal and astrocytic nuclei within the large cortical column datasets based on perinuclear fluorescence. These annotated nuclei were then used as seed points within their corresponding fluorescent channel for cell individualization in a highly interconnected network. For astrocytes, this technique provides the first method for characterization of complex morphology in an automated fashion over large areas without laborious dye filling or manual tracing

Reappraisal of transcallosal neuron organization in mice and evaluation of their dendritic remodeling and circuit integration following traumatic brain injury

Traumatic Brain Injury (TBI) is an enormous global socio-economic burden since, apart from its high death rate, it is the primary cause of coma worldwide and a prevalent cause of long-term disability. Until today there is no established treatment for dealing with the long-term outcomes of TBI despite many years of research. Although a lot is known about the pathophysiology of TBI in the damaged tissue and the surrounding area in case of focal lesion, only few studies have investigated the structural and functional integrity of the contralateral intact cortex. In order to explore this territory, this study employs a well-established and widely used animal model of focal open skull TBI known as the Controlled Cortical Impact (CCI) model. The first aim of this study was to systematically characterize a specific neuronal population, the transcallosal projection neurons, as they are the ones connecting the intact cortex with the lesioned cortex. The description of the organization of transcallosal neurons and their axonal projections at the contralateral hemisphere was carried out in healthy, non-injured C57Bl6 mice. Retrograde and anterograde tracing methods were implemented to label transcallosal cell bodies and their axonal projections, respectively. In addition, different injection coordinates were used in order to label transcallosal connections at distinct brain regions, including the motor cortex (M1), somatosensory cortex (S1), and barrel cortex, rostral and caudal to Bregma. In agreement with previous research, I observed that transcallosal projections are organized homotopically across the various brain regions, with the axonal terminals spanning the entire cortical column. Interestingly my study describes for the first time a non-negligible fraction of heterotopic transcallosal neurons that, in addition, display a slightly less strict layer distribution pattern compared to the homotopic ones. After the initial characterization of transcallosal neuron organization, I proceeded by investigating how these neurons with projections at the injury site are affected at various timepoints following focal TBI. I used GFPM mice to visualize dendrites and spines of transcallosal and non-transcallosal neurons, in order to examine their structural integrity at different timepoints post-injury. I detected significant differences in dendritic spine density and morphology between controls and injured mice, which were time-dependent. More specifically, the dendritic spine density in transcallosal neurons was strongly decreased as soon as 7days following injury. Interestingly, spine density in non-transcallosal neurons was not changed following TBI. In terms of spine shape, I found a morphological shift only for the apical tuft segments. These results point towards a general sensitivity of transcallosal spines to TBI-induced damage, where loss of spines (preferentially mature) seems to take place at 1-2 weeks post-injury and resolve at 3-6 weeks post-injury, indicative of late plasticity processes. As the anatomically connected neuronal population seems to recover overtime I then decided to further explore whether transcallosal circuit remodeling takes place after TBI. To do so I used the retrograde mono-trans-synaptic tracer SADΔG-GFP (EnvA) Rabies virus. In that way, I was able to distinctively label transcallosal neurons and their presynaptic partners and obtain an overview of the presynaptic population throughout the cortex across brain regions at different post-injury timepoints. This study demonstrates that spine plasticity did not result in adaptive circuit plasticity with the recruitment of other brain regions but rather that initial circuits were re-established. In brief, during this thesis I have demonstrated the adaptive plastic capacities of anatomically connected neurons to the brain injury. I believe that this knowledge may help in unraveling further compensatory plastic mechanisms that could then be therapeutically targeted to improve the outcome following brain injury

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

Studies on the function of PRG2/PLPPR3 in neuron morphogenesis

Neuron development follows a multifaceted sequence of cell migration, polarisation, neurite elongation, branching, tiling, and pruning. The implementation of this sequence differs between neuronal cell types and even in individual neurons between sub-compartments such as dendrites and axons. Membrane proteins are at a prime position in neurons to couple extrinsic morphogenetic signals with their intrinsic responses to orchestrate this defined morphological progression. The Phospholipid phosphatase-related / Plasticity-related gene (PLPPR/PRG)-family comprises five neuron-enriched and developmentally regulated membrane proteins with functions in cellular morphogenesis. At the start of this project, no publication had characterised the function of PLPPR3/PRG2 during neuron development. The presented work describes PLPPR3 as an axon-enriched protein localising to the plasma membrane and internal membrane compartments of neurons. Mutagenesis studies in cell lines establish the plasma membrane localisation of PLPPR3 as a regulator of its function to increase filopodia density (Chapter 2). Furthermore, the generation of a Plppr3-/- mouse line using CRISPR/Cas9 genome editing techniques (Chapter 3) enabled characterising endogenous phenotypes of PLPPR3 in neurons. In primary neuronal cultures, PLPPR3 was found to specifically control branch formation in a pathway with the phosphatase PTEN, without altering the overall growth capacity of neurons (Chapter 4). Loss of PLPPR3 specifically reduced branches forming from filopodia without affecting the stability of branches. This precise characterisation of PLPPR3 function unravelled the existence of parallel, independent programs for branching morphogenesis that are utilised and implemented differentially in developing axons and dendrites (Chapter 5). Furthermore, this thesis establishes multiple tools to study PLPPR3, the membrane lipid phosphatidylinositol-trisphosphate, and neuron morphogenesis by providing molecular tools, protocols, and semi-automated and automated image analysis pipelines (Appendix Chapter 7) and discusses experiments to test, refine and extend models of PLPPR3 function (Chapter 6). In summary, this thesis generated and utilised several tools and a Plppr3-/- mouse model to characterise PLPPR3 as a specific regulator of neuron branching morphogenesis. This precise characterisation refined and expanded the understanding of axon-specific branching morphogenesis.Nervenzellen entwickeln ihre komplexe Morphologie durch das Zusammenwirken diverser molekularer Entwicklungs-Programme der Zellkörper-Migration, der Polarisierung und der Morphogenese durch Wachstum, Verzweigung, Stabilisierung und Koordinierung ihrer Neuriten. Dabei unterscheidet sich die exakte Implementierung zwischen Nervenzell-Typen und selbst innerhalb einzelner Zellen zwischen Axonen und Dendriten. Diese unterschiedliche Morphogenese wird dabei speziell durch Membranproteine stark beeinflusst, die durch ihre Präsenz an der Plasmamembran Zell-extrinsische Signale mit den Zell-intrinsischen Morphogeneseprogrammen verbinden und beeinflussen. Die Familie der Phospholipid phosphatase-related / Plasticity-related gene (PLPPR/PRG) Proteine umfasst fünf Nervenzell-spezifische Membranproteine mit Effekten auf die Morphologie von Zellen. Zu Beginn dieses Projektes hatte noch keine Studie die Funktion des Familienmitglieds PLPPR3/PRG2 in Nervenzellen untersucht. Diese Dissertation beschreibt die Lokalisation von PLPPR3 an der Plasmamembran und in Zell-internen Membranstrukturen von Nervenzellen. Experimente in Zellkultur zeigen eine erhöhte Filopodien-Dichte nach Überexpression von PLPPR3, Mutagenese-Studien deuten eine strikte Kontrolle der Plasmamembran-Lokalisation an (Kapitel 2). Die Generierung einer Plppr3 Knockout Mauslinie mittels CRISPR/Cas9 Genom-Modifizierung (Kapitel 3) erlaubte eine Charakterisierung der endogenen Funktion von PLPPR3 in Nervenzellen. In Primärzellkultur von Nervenzellen des murinen Hippocampus zeigte sich, dass PLPPR3 im Zusammenspiel mit der Phosphatase PTEN spezifisch die Verzweigung von Nervenzellen kontrolliert, ohne deren Wachstumspotential global zu verändern (Kapitel 4). Dadurch kann PLPPR3 als ein Schalter zwischen Verzweigung und Verlängerung eines Nervenzell-Fortsatzes agieren. Der Verlust von PLPPR3 verursachte reduzierte spezifisch die Anzahl an Verzweigungen, die aus Filopodien entstanden, ohne dabei die Stabilität dieser Verzweigungen zu beeinflussen. Die präzise Charakterisierung dieser Funktion von PLPPR3 deckte auf, dass Verzweigungen von Nervenzell-Fortsätzen durch voneinander unabhängige Entwicklungsprogramme ausgebildet und stabilisiert werden können (Kapitel 5). Diese Programme werden von Axonen und Dendriten in unterschiedlicher Weise eingesetzt. Zusätzlich etabliert diese Arbeit sowohl diverse molekulare Werkzeuge und Visualisierungs-Protokolle zur Analyse von PLPPR3 und dem Membranlipid Phosphatidylinositol-Trisphosphat, als auch automatisierte Quantifizierungssoftware zur Studie der Nervenzellmorphologie (Appendix-Kapitel 7). Abschließend entwickelt und verfeinert die Dissertation mögliche Modelle zur PLPPR3-Funktion und zeigt experimentelle Strategien auf, um diese Modelle besser charakterisieren zu können (Kapitel 6). Zusammenfassend wurden in dieser Promotionsarbeit diverse Experimental- und Analyse-Strategien und eine Plppr3-/- Mauslinie entwickelt und genutzt, um PLPPR3 als einen spezifischen Regulator der Nervenzell-Morphogenese zu etablieren. Diese präzise Charakterisierung des PLPPR3 Phänotyps erlaubte zusätzlich eine Verfeinerung und Erweiterung der Erkenntnisse zur Axon-spezifischen Entwicklung von Verzweigungen

Nanoscale synaptic plasticity of aspiny interneuron dendrites with respect to characteristic fine structures

Long term plastic changes in synaptic function have been reported in GABAergic inhibitory interneurons in the mammalian hippocampus but structural changes associated with plasticity at the predominantly aspiny dendrites is not known. Aspiny dendrites are known to exhibit two distinct morphological types, smooth and varicose. Because of their rarity in neuropil we reimaged tissue in which long-term potentiation (LTP) had been induced by theta-burst stimulation (TBS) in acute hippocampal slices from mature rat using the tSEM method to obtain sufficiently large fields and measured the response of aspiny synapses. We report significant changes in synapse size after potentiation induction in the slice that is influenced by the type of fine structure on which it occurs. We developed quantitative methods to distinguish smooth from varicose aspiny dendrites and within varicose dendrites we found that the varicose and inter-varicose regions could respond differently to TBS. After potentiation induction in the slice synapse ultrastructure and mitochondria distribution changed in different directions in our two experiments but the relationship of synapse change and mitochondrial redistribution was consistent. In both experiments inter-varicose synapses significantly enlarged and were more likely to occur near mitochondria. In one experiment varicose synapses enlarged and were associated with more mitochondria. In the other synapses did not change but were less likely to associate with mitochondria. In smooth type synapses both experiments showed lower association with mitochondria and synapses either did not change or significantly decreased. Our findings of divergent ultrastructural changes among different aspiny dendrite regions may help to reconcile some of the disparate findings showing that similar stimulation protocols can lead to depressed or potentiated responses from inhibitory interneurons.Neuroscienc

The effect of spinal cord injury on vagal afferents.

Spinal cord injury (SCI) is a significant public health concern that leaves patients with a multitude of life-long disabilities. Major complications of SCI apart from paralysis, include deficits in bladder and bowel function. Lower urinary tract dysfunction continues to remain a top priority issue affecting quality of life for this population. The majority of visceral organs receive a dual sensory innervation from both spinal nerves as well as the vagus nerve. Following SCI, the vagus nerve is a potential pathway through which information from regions below the level of a spinal injury can travel directly to the brainstem, bypassing the spinal cord. The effect of SCI on the vagus nerve and the tissue it supplies has not been thoroughly examined. In order to advance bladder management after SCI, a thorough understanding of its neural control following chronic injury is needed to ultimately improve existing therapeutic options, as well as develop novel interventions that take advantage of this extraspinal route. The objective of this project was to describe the anatomical, neurochemical, and electrophysiological profiles of vagal innervation of the rat urinary bladder. Initially, the first study identified both single and double-labeled vagal afferents supplying the rat bladder and distal colon in the nodose ganglion (NG). The degree of neural innervation to the colon also was assessed, as a single axon that dichotomizes and innervates both organs can serve an important role for mediating both normal physiological and pathological reflexes. Following chronic SCI, we evaluated potential plasticity in subsets of NG neurons which contain projections that bypass the spinal cord from visceral organs, including those projections that specifically supply the bladder. Vagal sensory cell bodies displayed an increase in P2X3 expression and a decrease in IB4 binding, which also held true for many neurons innervating the bladder. Bladder-innervating neurons also displayed altered membrane electrophysiological properties, suggesting they are responsive to a chronic spinal injury. Even though SCI does not directly sever the vagus nerve, our results indicate vagal afferents, including those innervating the bladder, exhibit neurochemical plasticity post-injury that may have implications for visceral homeostatic mechanisms and nociceptive signaling