<meta charset="utf8"></head><body>使用 Neurolucida 360 进行树突状脊柱分析的方案

<ol>
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<table><tbody><tr><td>518F Immersion Oil</td><td>Zeiss</td><td>444960-0000-000</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM3050S</td><td>For slice preparation</td></tr><tr><td>Fine Forceps</td><td>FST</td><td>11150-10</td><td></td></tr><tr><td>Hemostat Forceps</td><td>FST</td><td>13020-12</td><td></td></tr><tr><td>Large Surgical Scissors</td><td>FST</td><td>14002-16</td><td></td></tr><tr><td>LSM 880 Confocal Microscope</td><td>Zeiss</td><td>LSM 880</td><td></td></tr><tr><td>Microscope Cover Glass</td><td>Fisherbrand</td><td>12-541-035</td><td></td></tr><tr><td>Mini-Peristaltic Pump II</td><td>Harvard Apparatus</td><td>70-2027</td><td>For perfusions</td></tr><tr><td>Neurolucida 360</td><td>MBF Bioscience</td><td>v2022.1.1</td><td>Spine Analysis Software</td></tr><tr><td>Neurolucida Explorer</td><td>MBF Bioscience</td><td>v2022.1.1</td><td>Spine Analysis Software</td></tr><tr><td>OCT Compound</td><td>Sakura Finetek</td><td>4583</td><td>For cryostat sectioning</td></tr><tr><td>Paraformaldehyde (37%)</td><td>Fisherbrand</td><td>F79-1</td><td></td></tr><tr><td>Plan-Apochromat 63x/1.40 Oil DIC</td><td>Zeiss</td><td>440762-9904-000</td><td></td></tr><tr><td>Scalpel Blade</td><td>FST</td><td>10022-00</td><td></td></tr><tr><td>Small Surgical Scissors</td><td>FST</td><td>14060-09</td><td></td></tr><tr><td>Spatula&amp;nbsp;</td><td>FST</td><td>10091-12</td><td></td></tr><tr><td>Sucrose</td><td>FIsherbrand</td><td>S5-500</td><td></td></tr><tr><td>Superfrost Plus Microslides</td><td>Diagger</td><td>ES4951+</td><td></td></tr><tr><td>Vectashield HardSet Mounting Medium</td><td>Vector Laboratories</td><td>H-1400-10</td><td></td></tr></tbody></table>

dendritic spine quantification using an automatic three-dimensional neuron reconstruction software

<meta charset="utf8"></head><body>作者聚焦:优化树突状棘分析，实现海马 CA1 顶端树突的平衡手动和自动评估

使用自动三维神经元重建软件进行树突状脊柱定量

Synaptic connections allow for the exchange and processing of information between neurons. The post-synaptic site of excitatory synapses is often formed ...

dendritic-spine-quantification-using-an-automatic-three-dimensional

Without Section

Research

Dendritic Spine Quantification Using an Automatic Three-Dimensional Neuron Reconstruction Software

JoVE Journal

Neuroscience

2.0K 次观看.  National Institutes of Health. 我们的研究范围是检查海马体树突棘的结构轮廓，特别是 CA-one 顶端树突。通过这种方式，我们试图回答围绕如何在细胞水平上编码同一树突不同部分的不同输入的问题。一个挑战是获得树突棘的高分辨率图像以进行脊柱分析。一旦你有了这些图像，以一种对研究人员来说不会过于繁重的方式最准确、最可靠地分析脊柱的方法就提出了额外的挑?...

视频: 作者聚焦:优化树突状棘分析，实现海马 CA1 顶端树突的平衡手动和自动评估

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

树突棘从锥体神经元的三维定量从人类诱导多能干细胞衍生

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

科研

发育生物学

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

在培养的中枢神经系统的神经元树突棘形态分析

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

神经科学

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

成像大鼠原代海马神经元树突棘使用结构照明显微镜

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

树突分支中的海马区对小鼠的齿状回评

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

3D Modeling of Dendritic Spines with Synaptic Plasticity

Computational modeling of diffusion and reaction of chemical species in a three-dimensional (3D) geometry is a fundamental method to understand the mechanisms of synaptic plasticity in dendritic spines. In this protocol, the detailed 3D structure of the dendrites and dendritic spines is modeled with meshes on the software Blender with CellBlender. The synaptic and extrasynaptic regions are defined on the mesh. Next, the synaptic receptor and synaptic anchor molecules are defined with their diffusion constants. Finally, the chemical reactions between synaptic receptors and synaptic anchors are included and the computational model is solved numerically with the software MCell. This method describes the spatiotemporal path of every single molecule in a 3D geometrical structure. Thus, it is very useful to study the trafficking of synaptic receptors in and out of the dendritic spines during the occurrence of synaptic plasticity. A limitation of this method is that the high number of molecules slows the speed of the simulations. Modeling of dendritic spines with this method allows the study of homosynaptic potentiation and depression within single spines and heterosynaptic plasticity between neighbor dendritic spines.

The protocol develops a three-dimensional (3D) model of a dendritic segment with dendritic spines for modeling synaptic plasticity. The constructed mesh can be used for computational modeling of AMPA receptor trafficking in the long-term synaptic plasticity using the software program Blender with CellBlender and MCell.

具有突触可塑性树突脊柱的三维建模

Computational modeling of diffusion and reaction of chemical species in a three-dimensional (3D) geometry is a fundamental method to understand the ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

脑切片和原细胞培养中金字塔神经元的弹道标记

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the morphology of processes and substructures within these neurons. As such, much of what is known about brain function has arisen alongside developments in imaging technologies that allow further insight into how neurons are organized and connected in the brain. Improvements in tissue clearing have allowed for high-resolution imaging of thick brain slices, facilitating morphological reconstruction and analyses of neuronal substructures, such as dendritic arbors and spines. In tandem, advances in image processing software provide methods of quickly analyzing large imaging datasets. This work presents a relatively rapid method of processing, visualizing, and analyzing thick slices of labeled neural tissue at high-resolution using CLARITY tissue clearing, confocal microscopy, and image analysis. This protocol will facilitate efforts toward understanding the connectivity patterns and neuronal morphologies that characterize healthy brains, and the changes in these characteristics that arise in diseased brain states.

Neuronal dendritic morphology often underlies function. Indeed, many disease processes that affect the development of neurons manifest with a morphological phenotype. This protocol describes a simple and powerful method for analyzing intact dendritic arbors and their associated spines.

通过 CLARITY 组织清除对完整神经元树突进行成像和定量

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the ...

Automatic Identification of Dendritic Branches and their Orientation

The structure of neuronal dendritic trees plays a key role in the integration of synaptic inputs in neurons. Therefore, characterization of the morphology of dendrites is essential for a better understanding of neuronal function. However, the complexity of dendritic trees, both when isolated and especially when located within neuronal networks, has not been completely understood. We developed a new computational tool, SOA (Segmentation and Orientation Analysis), which allows automatic measurement of the orientation of dendritic branches from fluorescence images of 2D neuronal cultures. SOA, written in Python, uses segmentation to distinguish dendritic branches from the image background and accumulates a database on the spatial direction of each branch. The database is then used to calculate morphological parameters such as the directional distribution of dendritic branches in a network and the prevalence of parallel dendritic branch growth. The data obtained can be used to detect structural changes in dendrites in response to neuronal activity and to biological and pharmacological stimuli.

Presented is a computational tool that allows simple and direct automatic measurement of orientations of neuronal dendritic branches from 2D fluorescence images.

树枝状分支及其方向的自动识别

The structure of neuronal dendritic trees plays a key role in the integration of synaptic inputs in neurons. Therefore, characterization of the morphology ...

3D Reconstruction and Analysis of Thin Subcellular Neuronal Structures using Focused-Ion Beam Scanning Electron Microscopy Data

Recent advances in scanning electron microscope technologies now permit the rapid three-dimensional (3D) analysis of ultrathin subcellular processes. Here, a methodological pipeline is presented to identify, visualize, and analyze thin neuronal processes, such as those that project into the presynaptic boutons of other neurons (termed 'spinules'). Using freely available software packages, this protocol demonstrates how to use a decision tree to identify common neuronal subcellular structures using morphological criteria within focused ion beam scanning electron microscopy (FIB-SEM) image volumes, with particular attention on identifying a diversity of spinules projecting into presynaptic boutons. In particular, this protocol describes how to trace spinules within neuronal synapses to produce 3D reconstructions of these thin subcellular projections, their parent neurites, and postsynaptic partners. Additionally, the protocol includes a list of freely available open-source software programs for analyzing FIB-SEM data and offers tips (e.g., smoothing, lighting) toward improving 3D reconstructions for visualization and publication. This adaptable protocol offers an entry point into the rapid nanoscale analysis of subcellular structures within FIB-SEM image volumes.

A flexible methodological pipeline to identify, visualize, and quantify thin subcellular neuronal processes within focused ion beam scanning electron microscopy image volumes using user-friendly open-source software packages.

Recent advances in scanning electron microscope technologies now permit the rapid three-dimensional (3D) analysis of ultrathin subcellular processes. ...

Rapid Golgi Stain for Dendritic Spine Visualization in Hippocampus and Prefrontal Cortex

Golgi impregnation, using the Golgi staining kit with minor adaptations, is used to impregnate dendritic spines in the rat hippocampus and medial prefrontal cortex. This technique is a marked improvement over previous methods of Golgi impregnation because the premixed chemicals are safer to use, neurons are consistently well impregnated, there is far less background debris, and for a given region, there are extremely small deviations in spine density between experiments. Moreover, brains can be accumulated after a certain point and kept frozen until further processing. Using this method any brain region of interest can be studied. Once stained and cover slipped, dendritic spine density is determined by counting the number of spines for a length of dendrite and expressed as spine density per 10 &#181;m dendrite.

The protocol describes a modification of the rapid Golgi method, which can be adapted to any part of the nervous system, for staining neurons in the hippocampus and medial prefrontal cortex of the rat.