Experience-dependent structural plasticity at pre- and postsynaptic sites of layer 2/3 cells in developing visual cortex

The developing brain can respond quickly to altered sensory experience by circuit reorganization. During a critical period in early life, neurons in the primary visual cortex rapidly lose responsiveness to an occluded eye and come to respond better to the open eye. While physiological and some of the molecular mechanisms of this process have been characterized, its structural basis, except for the well-known changes in the thalamocortical projection, remains obscure. To elucidate the relationship between synaptic remodeling and functional changes during this experience-dependent process, we used 2-photon microscopy to image synaptic structures of sparsely labeled layer 2/3 neurons in the binocular zone of mouse primary visual cortex. Anatomical changes at presynaptic and postsynaptic sites in mice undergoing monocular visual deprivation (MD) were compared to those in control mice with normal visual experience. We found that postsynaptic spines remodeled quickly in response to MD, with neurons more strongly dominated by the deprived eye losing more spines. These postsynaptic changes parallel changes in visual responses during MD and their recovery after restoration of binocular vision. In control animals with normal visual experience, the formation of presynaptic boutons increased during the critical period and then declined. MD affected bouton formation, but with a delay, blocking it after 3 d. These findings reveal intracortical anatomical changes in cellular layers of the cortex that can account for rapid activity-dependent plasticity

Multifaceted Changes in Synaptic Composition and Astrocytic Involvement in a Mouse Model of Fragile X Syndrome.

Fragile X Syndrome (FXS), a common inheritable form of intellectual disability, is known to alter neocortical circuits. However, its impact on the diverse synapse types comprising these circuits, or on the involvement of astrocytes, is not well known. We used immunofluorescent array tomography to quantify different synaptic populations and their association with astrocytes in layers 1 through 4 of the adult somatosensory cortex of a FXS mouse model, the FMR1 knockout mouse. The collected multi-channel data contained approximately 1.6 million synapses which were analyzed using a probabilistic synapse detector. Our study reveals complex, synapse-type and layer specific changes in the neocortical circuitry of FMR1 knockout mice. We report an increase of small glutamatergic VGluT1 synapses in layer 4 accompanied by a decrease in large VGluT1 synapses in layers 1 and 4. VGluT2 synapses show a rather consistent decrease in density in layers 1 and 2/3. In all layers, we observe the loss of large inhibitory synapses. Lastly, astrocytic association of excitatory synapses decreases. The ability to dissect the circuit deficits by synapse type and astrocytic involvement will be crucial for understanding how these changes affect circuit function, and ultimately defining targets for therapeutic intervention

Dense 4D nanoscale reconstruction of living brain tissue

Three-dimensional (3D) reconstruction of living brain tissue down to an individual synapse level would create opportunities for decoding the dynamics and structure–function relationships of the brain’s complex and dense information processing network; however, this has been hindered by insufficient 3D resolution, inadequate signal-to-noise ratio and prohibitive light burden in optical imaging, whereas electron microscopy is inherently static. Here we solved these challenges by developing an integrated optical/machine-learning technology, LIONESS (live information-optimized nanoscopy enabling saturated segmentation). This leverages optical modifications to stimulated emission depletion microscopy in comprehensively, extracellularly labeled tissue and previous information on sample structure via machine learning to simultaneously achieve isotropic super-resolution, high signal-to-noise ratio and compatibility with living tissue. This allows dense deep-learning-based instance segmentation and 3D reconstruction at a synapse level, incorporating molecular, activity and morphodynamic information. LIONESS opens up avenues for studying the dynamic functional (nano-)architecture of living brain tissue

A window on the ageing brain : Imaging synapses and their dynamics in vivo

The ageing process has an enormous impact on the human body and it represents a major risk factor for a number of diseases. Ageing is also associated with a progressive cognitive decline mainly involving the memory domain. Up to date many studies have investigated the structural and functional changes that occur in the ageing brain. Rather than neuronal loss, it is now widely accepted that synaptic impairments underlie the decreased cognitive performance. Such studies point to reduced synaptic density and plasticity, in specific brain regions, during ageing. However, most studies so far made use of either post-mortem or ex vivo preparations. Thus, the key question addressed in this thesis is to what extent synaptic elements are dynamic in the intact aged brain. A combination of in vivo two-photon imaging, correlated two-photon-electron microscopy and novel computational tools was used to study synaptic boutons in the aged mouse somatosensory cortex. Unexpectedly, circuit-specific increased rates of bouton formation, elimination, and destabilization were found. Age related increased dynamics greatly affected large (i.e., strong) boutons, thought to encode long-term memory, as opposed to smaller ones. The rigorous measurement of the size and location of axonal boutons, achieved for the first time in vivo, showed that while the average density and size of boutons was not affected by ageing, bouton size changes were greater in the aged animals. Such increased size fluctuations were again confined to larger, persistent boutons., Long-term memory impairment, as assessed in a novel behavioural task, was therefore associated with increased, rather than decreased, synaptic destabilization and dynamics, suggesting the existence of a novel mechanism underlying age related cognitive decline.Open Acces

Enabling Scalable Neurocartography: Images to Graphs for Discovery

In recent years, advances in technology have enabled researchers to ask new questions predicated on the collection and analysis of big datasets that were previously too large to study. More specifically, many fundamental questions in neuroscience require studying brain tissue at a large scale to discover emergent properties of neural computation, consciousness, and etiologies of brain disorders. A major challenge is to construct larger, more detailed maps (e.g., structural wiring diagrams) of the brain, known as connectomes. Although raw data exist, obstacles remain in both algorithm development and scalable image analysis to enable access to the knowledge within these data volumes. This dissertation develops, combines and tests state-of-the-art algorithms to estimate graphs and glean other knowledge across six orders of magnitude, from millimeter-scale magnetic resonance imaging to nanometer-scale electron microscopy. This work enables scientific discovery across the community and contributes to the tools and services offered by NeuroData and the Open Connectome Project. Contributions include creating, optimizing and evaluating the first known fully-automated brain graphs in electron microscopy data and magnetic resonance imaging data; pioneering approaches to generate knowledge from X-Ray tomography imaging; and identifying and solving a variety of image analysis challenges associated with building graphs suitable for discovery. These methods were applied across diverse datasets to answer questions at scales not previously explored

Three-dimensional reengineering of neuronal microcircuits : The cortical column in silico

The presented thesis will describe a pipeline to reengineer three-dimensional, anatomically realistic, functional neuronal networks with subcellular resolution. The pipeline consists of five methods: 1. "NeuroCount" provides the number and three-dimensional distribution of all neuron somata in large brain regions. 2. "NeuroMorph" provides authentic neuron tracings, comprising dendrite and axon morphology. 3. "daVinci" registers the neuron morphologies to a standardized reference framework. 4. "NeuroCluster" objectively groups the standardized tracings into anatomical neuron types. 5. "NeuroNet" combines the number and distribution of neurons and neuron-types with the standardized tracings and determines the neuron-type- and position-specific number of synaptic connections for any two types of neuron. The developed methods are demonstrated by reengineering the thalamocortical lemniscal microcircuit in the somatosensory system of rats. There exists an one-to-one correspondence between the sensory information obtained by a single facial whisker and segregated areas in the thalamus and cortex. The reengineering of this pathway results in a column-shaped network model of ~15200 excitatory full-compartmental cortical neurons. This network is synaptically connected to ~285 pre-synaptic thalamic neurons. Animation of this "cortical column in silico" with measured physiological input will help to gain a mechanistic understanding of neuronal sensory information processing in the mammalian brain

Towards a comprehensive understanding of brain machinery by correlative microscopy.

Unraveling the complexity of brain structure and function is the biggest challenge of contemporary science. Due to their flexibility, optical techniques are the key to exploring this intricate network. However, a single imaging technique can reveal only a small part of this machinery due to its inherent multilevel organization. To obtain a more comprehensive view of brain functionality, complementary approaches have been combined. For instance, brain activity was monitored simultaneously on different spatiotemporal scales with functional magnetic resonance imaging and calcium imaging. On the other hand, dynamic information on the structural plasticity of neuronal networks has been contextualized in a wider framework combining two-photon and light-sheet microscopy. Finally, synaptic features have been revealed on previously in vivo imaged samples by correlative light-electron microscopy. Although these approaches have revealed important features of brain machinery, they provided small bridges between specific spatiotemporal scales, lacking an omni-comprehensive view. In this perspective, we briefly review the state of the art of correlative techniques and propose a wider methodological framework fusing multiple levels of brain investigation

Pharmacological BACE1 inhibitor treatment during early progression of β-amyloid pathology maximizes therapeutic efficacy

Alzheimer’s disease (AD) is a chronic neurodegenerative disease of the central nervous system (CNS) characterized by progressive cognitive decline. AD is the most common cause of all dementia cases worldwide, and as a result of demographic aging the number of affected individuals grows at an alarming rate. The amyloid hypothesis of Alzheimer’s disease (AD) emphasizes amyloid-β peptide (Aβ) as primary cause of the disease, with toxic effects on synapses leading to cognitive decline and memory impairments. Beta site amyloid precursor protein cleaving enzyme 1 (BACE1) as the rate-limiting enzyme of amyloidogenic processing of amyloid precursor protein (APP), is one of the prime drug targets for the treatment of AD. However, despite the development of potent and selective small-molecule BACE1 inhibitors, so far all human clinical trials have failed to rescue the cognitive decline in AD patients. Recent findings indicate that treatment has to be commenced before AD symptoms arise, since in symptomatic patients β amyloid deposition has already reached a plateau. Moreover, several studies have described dose-dependent adverse effects in animal models. Therefore, it is a central requirement to develop a treatment strategy that is therapeutically effective and at the same time avoids excessive interference with physiological function of BACE1. In this study, transgenic AD mice were treated at an early stage of β amyloid pathology with the potent, blood brain barrier penetrating BACE1 inhibitor NB-360. Longitudinal in vivo two-photon imaging was performed to repeatedly monitor individual amyloid plaques, presynaptic boutons and axonal dystrophies in living mice. In APPPS1 mice pharmacological BACE1 inhibition fails to revert but significantly reduces the progressive amyloid deposition and mitigates presynaptic pathology. Notably, the data show that plaque seed formation, rather than the subsequent phase of gradual plaque growth, is most sensitive to BACE1 inhibition. These results imply, that preventive BACE1 inhibitor treatment is required to achieve therapeutic efficacy. For clinical therapy, to exploit the particular susceptibility of plaque formation to BACE1 inhibition, a dosage has to be empirically determined that effectively halts formation of new plaques rather than aiming at halting plaque growth. This strategy might optimally balance potential mechanism-based adverse effects and efficacious reduction of β amyloid deposition.Morbus Alzheimer ist eine chronische neurodegenerative Erkrankung des zentralen Nervensystems und äußert sich in progressivem Verlust kognitiver Funktionen und Gedächtnisleistung. Die Erkrankung ist die weltweit häufigste Ursache für Demenz und aufgrund demografischer Alterung in den Industrie¬ländern, nimmt die Zahl der Alzheimer Patienten stetig zu. Der Amyloid-Kaskaden-Hypothese zufolge, wird die Alzheimer Erkrankung durch pathologische Akkumulation und Aggregation des Aβ-Peptids (Aβ) ausgelöst. Aβ wird durch sequentielle enzymatische Spaltung des Amyloid-Vorläufer-proteins APP produziert. Die β-Sekretase BACE1 initiiert den ersten Schritt dieses sogenannten amyloiden Prozessierungswegs und ist somit eines der aussichtsreichsten Wirkstoffziele zur Senkung des Aβ-Spiegels. Im Verlauf der letzten Jahre wurden sehr wirksame und zugleich selektive BACE1 Inhibitoren hergestellt, doch bislang sind klinische Studien daran gescheitert, den progressiven Gedächtnisverlust aufzuhalten. Neueste Erkenntnisse weisen darauf hin, dass die Behandlung bereits vor dem Auftreten der ersten Symptome begonnen werden muss, da in symptomatischen Patienten die Ablagerung von Aβ in den meisten Fällen bereits abgeschlossen ist. Hinzu kommt, dass in den letzten Jahren vermehrt negative Begleiterscheinungen der Behandlung mit BACE1 Inhibitoren in Mäusen bekannt geworden sind. Die entscheidende Herausforderung ist somit, eine Behandlungsstrategie zu entwickeln, welche einerseits die physiologische Funktion von BACE1 nicht zu stark beeinträchtigt, aber zugleich therapeutische Effizienz gewährleistet. In der vorliegenden Studie wurden transgene Alzheimer Mäuse in einem frühen Stadium der β amyloiden Pathologie mit dem potenten BACE1 Inhibitor NB-360 behandelt. Mittels chronischer in vivo Mikroskopie konnten einzelne β amyloide Plaques, präsynaptische Boutons und axonale Dystrophien in lebenden Mäusen verfolgt werden. Die Behandlung erbrachte zwar keinen Rückgang der Aβ Ablagerung, konnte jedoch deren Fortschreiten verringern, sowie die progressive axonale Pathologie abschwächen. Insbesondere zeigten unsere Daten, dass die BACE1 Inhibitor Behandlung einen wesentlich größeren Einfluss auf die Bildung neuer β amyloider Plaques, als auf deren Wachstum hatte. Diese Ergebnisse weisen darauf hin, dass die Behandlung mit BACE1 Inhibitoren präventiv erfolgen muss. Für die klinische Anwendung könnte man sich die besondere Anfälligkeit der Neubildung von Plaques zu Nutze machen und über empirische Versuche einen Dosisbereich bestimmen, welcher ausreicht, die Neubildung von Plaques zu unterdrücken. Diese Strategie könnte zu einer ausgewogenen Behandlung führen, welche die progressive Aβ Ablagerung verzögert und gleichermaßen das Auftreten von Nebenwirkungen verhindert

살아있는 뉴런과 동물에서 mRNA 관찰에 대한 연구

학위논문(박사) -- 서울대학교대학원 : 자연과학대학 물리·천문학부(물리학전공), 2021.8. 이병훈.mRNA는 유전자 발현의 첫번째 산물이면서, 리보솜과 함께 단백질을 합성한다. 특히 뉴런에서, 몇몇 RNA들은 자극에 의해 만들어지고, 뉴런의 특정 부분으로 수송되어 국소적으로 단백질 양을 조절할 수 있게 한다. 최근 mRNA 표지 기술의 발전으로 살아있는 세포에서 단일 mRNA를 관찰하는 것이 가능해졌다. 이 연구에서, 우리는 RNA 이미징 기술을 이용해, 기억 형성과 상기할 때 활성화된 뉴런의 집합을 찾는 것 뿐 아니라, 뉴런의 축삭돌기에서 mRNA가 어떻게 수송되는지를 관찰했다. 이 논문의 첫 부분에서 우리는 신경 자극에 반응해서 만들어지는 것으로 알려진, Arc 유전자의 전사를 관찰하였다. 기억은 engram 혹은 기억 흔적 (memory trace)라고 불리는 뉴런들의 집합에 저장되어 있다고 생각된다. 그러나, 시간에 따라서 이런 기억 흔적세포들의 집합이 어떻게 변하고, 변화하면서도 어떻게 정보를 유지할 수 있는지 잘 알려져 있지 않다. 또한, 살아있는 동물에서, 기억 흔적세포를 긴 시간 동안 여러 번 찾아내는 것은 어려운 일이었다. 이 연구에서는 genetically-encoded RNA indicator (GERI) 기술을 사용해, 기억 흔적세포의 표식으로 널리 사용되는 Arc mRNA의 전사과정을 살아있는 쥐에서 관찰하였다. GERI를 이용함으로써, 기존 방법들의 한계점이었던 시간 제약 없이, 실시간으로 Arc를 발현하는 뉴런들을 찾아낼 수 있었다. 쥐에게 공간 공포 기억을 주고 나서 여러 번 기억을 상기시키는 행동실험 후에 Arc를 발현하는 세포를 식별했을 때, CA1에서는 Arc를 발현하는 세포가 이틀 후에는 더 이상 활성화되지 않았으나, RSC의 경우 4퍼센트의 뉴런들이 계속해서 활성화하는 것을 관찰했다. 신경활동과 유전자 발현을 같이 조사하기 위해, 쥐가 가상 환경을 탐험하고 있을 때 GERI와 칼슘 이미징을 동시에 진행하였다. 그 결과, 기억을 형성할 때와 상기시킬 때 Arc를 발현했던 뉴런들이 기억을 표상하는 것을 알아낼 수 있었다. 이처럼 GERI 기술을 이용해 살아있는 동물에서 유전자 발현된 세포를 찾아내는 방식은 다양한 학습 및 기억 과정에서 기억 흔적세포의 dynamics에 대해 알아낼 수 있을 것으로 기대된다. 이 논문의 두번째 부분에서, 우리는 세포 골격의 기본 구성 단위가 되는 β-actin의 mRNA를 축삭돌기에서 관찰하였다. mRNA의 국소화 (localization)를 통한 국소 단백질 합성은 축삭돌기 (axon)의 성장과 재생에 중요한 역할이 있다고 알려져 있다. 하지만, 아직 mRNA의 국소화가 축삭돌기에서 어떻게 조절되고 있는지 잘 알려져 있지 않다. 이 문제를 해결하기 위해서, 우리는 모든 β-actin mRNA가 형광으로 표지된 유전자 변형 쥐를 이용해, 살아있는 축삭돌기에서 β-actin mRNA를 관찰하였다. 이 쥐의 뉴런을 축삭을 구분해 줄 수 있는 미세유체 장치 (microfluidic device)에 배양한 뒤에, β-actin mRNA를 관찰하고 추적을 진행했다. 축삭은 세포 몸통으로부터 길게 자라기 때문에 mRNA가 먼 거리를 수송되어야 함에도 불구하고, 대부분의 mRNA가 수상돌기에 비해 덜 움직이고 작은 영역에서 움직이는 것을 보았다. 우리는 β-actin mRNA가 주로 축삭돌기의 가지가 될 수 있는 filopodia 근처와, 시냅스가 만들어지는 bouton 근처에 국소화되는 것을 관찰했다. Filopodia와 bouton이 actin이 풍부한 부분으로 알려져 있기 때문에, 우리는 액틴 필라멘트와 β-actin mRNA의 움직임간에 연관성을 조사했다. 흥미롭게도, 우리는 β-actin mRNA가 액틴 필라멘트와 같이 국소화 되고, β-actin mRNA가 액틴 필라멘트 안에서 sub-diffusive한 움직임을 보였으며, 먼 거리를 움직이던 mRNA도 액틴 필라멘트에 고정되는 모습도 확인할 수 있었다. 축삭에서 β-actin mRNA 움직임을 본 이번 관찰은 mRNA 수송 및 국소화에 대한 생물물리학 적 메커니즘의 기반이 될 수 있을 것이다.mRNA is the first product of the gene expression and facilitates the protein synthesis. Especially in neurons, some RNAs are transcribed in response to stimuli and transported to the specific region, altering local proteome for neurons to function normally. Recent advances of mRNA labeling techniques allowed us to observe the single mRNAs in live cells. In this thesis, we applied RNA imaging technique not only to identify the neuronal ensemble that activated during memory formation and retrieval, but also to traffic mRNAs transported to the axon. In the first part of the thesis, we observed the transcription site of Arc gene, one of the immediate-early gene, which is rapidly transcribed upon the neural stimuli. Because of the characteristic of expressing in response to stimuli, Arc is widely used as a marker for memory trace cells thought to store memories. However, little is known about the ensemble dynamics of these cells because it has been challenging to observe them repeatedly over long periods of time in vivo. To overcome this limitation, we present a genetically-encoded RNA indicator (GERI) technique for intravital chronic imaging of endogenous Arc mRNA. We used our GERI to identify Arc-positive neurons in real time without the time lag associated with reporter protein expression in conventional approaches. We found that Arc-positive neuronal populations rapidly turned over within two days in CA1, whereas ~4% of neurons in the retrosplenial cortex consistently expressed Arc upon contextual fear conditioning and repeated memory retrievals. Dual imaging of GERI and calcium indicator in CA1 of mice navigating a virtual reality environment revealed that only the overlapping population of neurons expressing Arc during encoding and retrieval exhibited relatively high calcium activity in a context-specific manner. This in vivo RNA imaging approach has potential to unravel the dynamics of engram cells underlying various learning and memory processes. In the second part of this thesis, we imaged β-actin mRNAs, which can generate a cytoskeletal protein, β-actin, through translation. Local protein synthesis has a critical role in axonal guidance and regeneration. Yet it is not clearly understood how the mRNA localization is regulated in axons. To address these questions, we investigated mRNA motion in live axons using a transgenic mouse that expresses fluorescently labeled endogenous β-actin mRNA. By culturing hippocampal neurons in a microfluidic device that allows separation of axons from dendrites, we performed single particle tracking of β-actin mRNA selectively in axons. Although axonal mRNAs need to travel a long distance, we observed that most axonal mRNAs show much less directed motion than dendritic mRNAs. We found that β-actin mRNAs frequently localize at the neck of filopodia which can grow as axon collateral branches and at varicosities where synapses typically occur. Since both filopodia and varicosities are known as actin-rich areas, we investigated the dynamics of actin filaments and β-actin mRNAs simultaneously by using high-speed dual-color imaging. We found that axonal mRNAs colocalize with actin filaments and show sub-diffusive motion within the actin-rich regions. The novel findings on the dynamics of β-actin mRNA will shed important light on the biophysical mechanisms of mRNA transport and localization in axons.1. INTRODUCTION, 1 1.1. Neuronal ensemble, 1 1.2. Immediate-early Gene (IEG), 3 1.3. Methods for IEG-positive neurons, 3 1.4. Two-photon microscope, 5 1.5. References, 7 2. IMAGING ARC mRNA TRANSCRIPTION SITES IN LIVE MICE, 9 2.1. Introduction, 9 2.2. Materials and Methods, 10 2.3. Results and Discussion, 18 2.4. References, 26 3. NEURONS EXPRESSING ARC mRNA DURING REPEATED MEMORY RETRIEVALS, 28 3.1. Introduction, 28 3.2. Results and Discussion, 28 3.3. References, 35 4. NEURAL ACTIVITIES OF ARC+ NEURONS, 36 4.1. Introduction, 36 4.2. Materials and Methods, 37 4.3. Results and Discussion, 38 4.4. References, 52 5. AXONAL mRNA DYNAMICS IN LIVE NEURONS, 54 5.1. Introduction, 54 5.2. Materials and Methods, 55 5.3. Results and Discussion, 59 5.4. References, 70 6. CONCLUSION AND OUTLOOK, 72 ABSTRACT IN KOREAN (국문초록), 76박