Current Protocols in Neuroscience最新文献_第2页

Expansion Microscopy for Beginners: Visualizing Microtubules in Expanded Cultured HeLa Cells 初学者扩展显微镜:在扩展培养的HeLa细胞中可视化微管

Current Protocols in Neuroscience Pub Date : 2020-06-04 DOI: 10.1002/cpns.96

Chi Zhang, Jeong Seuk Kang, Shoh M. Asano, Ruixuan Gao, Edward S. Boyden

引用次数: 15

Visual Psychophysics in Head-Fixed Mice 头部固定小鼠的视觉心理物理学

Current Protocols in Neuroscience Pub Date : 2020-03-26 DOI: 10.1002/cpns.95

Richard J. Krauzlis, Nick Nichols, Krsna V. Rangarajan, Kerry McAlonan, Sheridan Goldstein, Daniel Yochelson, Lupeng Wang

{"title":"Visual Psychophysics in Head-Fixed Mice","authors":"Richard J. Krauzlis, Nick Nichols, Krsna V. Rangarajan, Kerry McAlonan, Sheridan Goldstein, Daniel Yochelson, Lupeng Wang","doi":"10.1002/cpns.95","DOIUrl":"10.1002/cpns.95","url":null,"abstract":"We describe a set of protocols for doing visual psychophysical experiments in head-fixed mice. The goal of this approach was to conduct in mice the same type of precise and well-controlled tests of visual perception and decision making as is commonly done in primates. For example, these experimental protocols were the basis for our demonstration that mice are capable of visual selective attention in paradigms adapted from classic attention cueing paradigms in primates. Basic Protocol 1 describes how to construct the experimental apparatus, including the removable wheel assembly on which the mice run during the visual tasks, the lick spout used to deliver rewards and detect licks, and the behavioral box that places these components together with the visual displays. We also describe the functions of the computerized control system and the design of the customized head fixture. Basic Protocol 2 describes the preparation of mice for the experiments, including the detailed surgical steps. Basic Protocol 3 describes the transition to a food schedule for the mice and how to operate the experimental apparatus. Basic Protocol 4 outlines the logic of the task design and the steps necessary for training the mice. Finally, Basic Protocol 5 describes how to obtain and analyze the psychometric data. Our methods include several distinctive features, including a custom quick-release method for holding the head and specific strategies for training mice over multiple weeks. Published 2020. U.S. Government.Basic Protocol 1: Experimental apparatusBasic Protocol 2: Head fixture surgeryBasic Protocol 3: General operation of the experimental apparatusBasic Protocol 4: Behavioral task design and trainingBasic Protocol 5: Psychometric data collection and analysis","PeriodicalId":40016,"journal":{"name":"Current Protocols in Neuroscience","volume":"92 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpns.95","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37775198","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 7

Ex Vivo Imaging of Mitochondrial Dynamics and Trafficking in Astrocytes 星形胶质细胞线粒体动力学和运输的离体成像

Current Protocols in Neuroscience Pub Date : 2020-03-16 DOI: 10.1002/cpns.94

Julia K. Farnan, Kayla K. Green, Joshua G. Jackson

{"title":"Ex Vivo Imaging of Mitochondrial Dynamics and Trafficking in Astrocytes","authors":"Julia K. Farnan, Kayla K. Green, Joshua G. Jackson","doi":"10.1002/cpns.94","DOIUrl":"10.1002/cpns.94","url":null,"abstract":"Mitochondria are essential organelles involved in energy supply and calcium homeostasis. The regulated distribution of mitochondria in polarized cells, particularly neurons, is thought to be essential to these roles. Altered mitochondrial function and impairment of mitochondrial distribution and dynamics is implicated in a number of neurologic disorders. Several recent reports have described mechanisms regulating the activity-dependent distribution of mitochondria within astrocyte processes and the functional consequences of altered mitochondrial transport. Here we provide an ex vivo method for monitoring the transport of mitochondria within the processes of astrocytes using organotypic “slice” cultures. These methods can be easily adapted to investigate a wide range of mitochondrial behaviors, including fission and fusion dynamics, mitophagy, and calcium signaling in astrocytes and other cell types of the central nervous system. © 2020 by John Wiley & Sons, Inc.Basic Protocol 1: Preparation of brain slicesBasic Protocol 2: Preparation of gene gun bulletsBasic Protocol 3: Gene gun transfection of slicesBasic Protocol 4: Visualization and tracking of mitochondrial movementAlternate Protocol: Transduction of EGFP-mito via viral injection of the neonatal mouse brain","PeriodicalId":40016,"journal":{"name":"Current Protocols in Neuroscience","volume":"92 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-03-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpns.94","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37741789","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Translatome Analyses Using Conditional Ribosomal Tagging in GABAergic Interneurons and Other Sparse Cell Types 条件核糖体标记在gaba能中间神经元和其他稀疏细胞类型中的翻译组分析

Current Protocols in Neuroscience Pub Date : 2020-03-05 DOI: 10.1002/cpns.93

Vivek Mahadevan, Areg Peltekian, Chris J. McBain

{"title":"Translatome Analyses Using Conditional Ribosomal Tagging in GABAergic Interneurons and Other Sparse Cell Types","authors":"Vivek Mahadevan, Areg Peltekian, Chris J. McBain","doi":"10.1002/cpns.93","DOIUrl":"10.1002/cpns.93","url":null,"abstract":"GABAergic interneurons comprise a small but diverse subset of neurons in the mammalian brain that tightly regulate neuronal circuit maturation and information flow and, ultimately, behavior. Because of their centrality in the etiology of numerous neurological disorders, examining the molecular architecture of these neurons under different physiological scenarios has piqued the interest of the broader neuroscience community. The last few years have seen an explosion in next-generation sequencing (NGS) approaches aimed at identifying genetic and state-dependent subtypes in neuronal diversity. Although several approaches are employed to address neuronal molecular diversity, ribosomal tagging has emerged at the forefront of identifying the translatomes of neuronal subtypes. This approach primarily relies on Cre recombinase–driven expression of hemagglutinin A (HA)–tagged RiboTag mice exclusively in the neuronal subtype of interest. This allows the immunoprecipitation of cell-type-specific, ribosome-engaged mRNA, expressed both in the soma and the neuronal processes, for targeted quantitative real-time PCR (qRT-PCR) or high-throughput RNA sequencing analyses. Here we detail the typical technical caveats associated with successful application of the RiboTag technique for analyzing GABAergic interneurons, and in theory other sparse cell types, in the central nervous system. Published 2020. U.S. Government.Basic Protocol 1: Breeding mice to obtain RiboTag homozygositySupport Protocol 1: Detection of ectopic Cre recombinase expressionBasic Protocol 2: The RiboTag assaySupport Protocol 2: Real-time quantitative PCR (qRT-PCR) assay of RiboTag-derived cell-type-specific RNASupport Protocol 3: Construction of cell-type-specific RNA-seq librarySupport Protocol 4: Secondary analyses of RiboTag-derived RNA-seq dataset","PeriodicalId":40016,"journal":{"name":"Current Protocols in Neuroscience","volume":"92 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpns.93","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38089333","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 3

High-Resolution Three-Dimensional Imaging of Individual Astrocytes Using Confocal Microscopy. 利用共聚焦显微镜对单个星形胶质细胞进行高分辨率三维成像。

Current Protocols in Neuroscience Pub Date : 2020-03-01 DOI: 10.1002/cpns.92

Anze Testen, Ronald Kim, Kathryn J Reissner

{"title":"High-Resolution Three-Dimensional Imaging of Individual Astrocytes Using Confocal Microscopy.","authors":"Anze Testen, Ronald Kim, Kathryn J Reissner","doi":"10.1002/cpns.92","DOIUrl":"https://doi.org/10.1002/cpns.92","url":null,"abstract":"Astrocytes play numerous vital roles in the central nervous system. Accordingly, it is of merit to identify structural and functional properties of astrocytes in both health and disease. The majority of studies examining the morphology of astrocytes have employed immunoassays for markers such as glial fibrillary acidic protein, which are insufficient to encapsulate the considerable structural complexity of these cells. Herein, we describe a method utilizing a commercially available and validated, genetically encoded membrane-associated fluorescent marker of astrocytes, AAV5-GfaABC1D-Lck-GFP. This tool and approach allow for visualization of a single isolated astrocyte in its entirety, including fine peripheral processes. Astrocytes are imaged using confocal microscopy and reconstructed in three dimensions to obtain detailed morphometric data. We further provide an immunohistochemistry procedure to assess colocalization of isolated astrocytes with synaptic markers throughout the z-plane. This technique, which can be utilized via a standard laboratory confocal microscope and Imaris software, allows for detailed analysis of the morphology and synaptic colocalization of astrocytes in fixed tissue. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Microinjection of AAV5-GfaABC1D-Lck-GFP into the nucleus accumbens of rats Basic Protocol 2: Tissue processing and immunohistochemistry for post-synaptic density-95 Basic Protocol 3: Single-cell image acquisition Basic Protocol 4: Three-dimensional reconstruction of single cells Basic Protocol 5: Three-dimensional colocalization analysis.","PeriodicalId":40016,"journal":{"name":"Current Protocols in Neuroscience","volume":"91 1","pages":"e92"},"PeriodicalIF":0.0,"publicationDate":"2020-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpns.92","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37653639","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 18

Assessing Neuron-Astrocyte Spatial Interactions Using the Neuron-Astrocyte Proximity Assay. 利用神经元-星形胶质细胞接近试验评估神经元-星形胶质细胞空间相互作用。

Current Protocols in Neuroscience Pub Date : 2020-03-01 DOI: 10.1002/cpns.91

Aina Badia-Soteras, J Christopher Octeau, Mark H G Verheijen, Baljit S Khakh

{"title":"Assessing Neuron-Astrocyte Spatial Interactions Using the Neuron-Astrocyte Proximity Assay.","authors":"Aina Badia-Soteras, J Christopher Octeau, Mark H G Verheijen, Baljit S Khakh","doi":"10.1002/cpns.91","DOIUrl":"10.1002/cpns.91","url":null,"abstract":"Astrocytes are morphologically complex cells with numerous close contacts with neurons at the level of their somata, branches, and branchlets. The smallest astrocyte processes make discrete contacts with synapses at scales that cannot be observed by standard light microscopy. At such contact points, astrocytes are thought to perform both homeostatic and neuromodulatory roles-functions that are proposed to be determined by their close spatial apposition. To study such spatial interactions, we previously developed a Förster resonance energy transfer (FRET)-based approach, which enables observation and tracking of the static and dynamic proximity of astrocyte processes with synapses. The approach is compatible with standard imaging techniques such as confocal microscopy and permits assessment of the most proximate contacts between astrocytes and neurons in live tissues. In this protocol article we describe the approach to analyze the contacts between striatal astrocyte processes and corticostriatal neuronal projection terminals onto medium spiny neurons. We report the required protocols in detail, including adeno-associated virus microinjections, acute brain slice preparation, imaging, and post hoc FRET quantification. The article provides a detailed description that can be used to characterize and study astrocyte process proximity to synapses in living tissue. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Förster resonance energy transfer imaging in cultured cells Basic Protocol 2: Förster resonance energy transfer imaging with the neuron-astrocyte proximity assay in acute brain slices.","PeriodicalId":40016,"journal":{"name":"Current Protocols in Neuroscience","volume":"91 1","pages":"e91"},"PeriodicalIF":0.0,"publicationDate":"2020-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpns.91","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37652777","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 5

Back and to the Future: From Neurotoxin-Induced to Human Parkinson's Disease Models. 回到未来:从神经毒素诱导到人类帕金森病模型。

Current Protocols in Neuroscience Pub Date : 2020-03-01 DOI: 10.1002/cpns.88

Mikko Airavaara, Ilmari Parkkinen, Julia Konovalova, Katrina Albert, Piotr Chmielarz, Andrii Domanskyi

{"title":"Back and to the Future: From Neurotoxin-Induced to Human Parkinson's Disease Models.","authors":"Mikko Airavaara, Ilmari Parkkinen, Julia Konovalova, Katrina Albert, Piotr Chmielarz, Andrii Domanskyi","doi":"10.1002/cpns.88","DOIUrl":"https://doi.org/10.1002/cpns.88","url":null,"abstract":"Parkinson's disease (PD) is an age-related neurodegenerative disorder characterized by motor symptoms such as tremor, slowness of movement, rigidity, and postural instability, as well as non-motor features like sleep disturbances, loss of ability to smell, depression, constipation, and pain. Motor symptoms are caused by depletion of dopamine in the striatum due to the progressive loss of dopamine neurons in the substantia nigra pars compacta. Approximately 10% of PD cases are familial arising from genetic mutations in α-synuclein, LRRK2, DJ-1, PINK1, parkin, and several other proteins. The majority of PD cases are, however, idiopathic, i.e., having no clear etiology. PD is characterized by progressive accumulation of insoluble inclusions, known as Lewy bodies, mostly composed of α-synuclein and membrane components. The cause of PD is currently attributed to cellular proteostasis deregulation and mitochondrial dysfunction, which are likely interdependent. In addition, neuroinflammation is present in brains of PD patients, but whether it is the cause or consequence of neurodegeneration remains to be studied. Rodents do not develop PD or PD-like motor symptoms spontaneously; however, neurotoxins, genetic mutations, viral vector-mediated transgene expression and, recently, injections of misfolded α-synuclein have been successfully utilized to model certain aspects of the disease. Here, we critically review the advantages and drawbacks of rodent PD models and discuss approaches to advance pre-clinical PD research towards successful disease-modifying therapy. © 2020 The Authors.","PeriodicalId":40016,"journal":{"name":"Current Protocols in Neuroscience","volume":"91 1","pages":"e88"},"PeriodicalIF":0.0,"publicationDate":"2020-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpns.88","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37635623","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 28

Assaying Fear Memory Discrimination and Generalization: Methods and Concepts 分析恐惧记忆、辨析与概化:方法与概念

Current Protocols in Neuroscience Pub Date : 2020-01-29 DOI: 10.1002/cpns.89

Hadley C. Bergstrom

{"title":"Assaying Fear Memory Discrimination and Generalization: Methods and Concepts","authors":"Hadley C. Bergstrom","doi":"10.1002/cpns.89","DOIUrl":"10.1002/cpns.89","url":null,"abstract":"Generalization describes the transfer of conditioned responding to stimuli that perceptually differ from the original conditioned stimulus. One arena in which discriminant and generalized responding is of particular relevance is when stimuli signal the potential for harm. Aversive (fear) conditioning is a leading behavioral model for studying associative learning and memory processes related to threatening stimuli. This article describes a step-by-step protocol for studying discrimination and generalization using cued fear conditioning in rodents. Alternate conditioning paradigms, including context generalization, differential generalization, discrimination training, and safety learning, are also described. The protocol contains instructions for constructing a cued fear memory generalization gradient and methods for isolating discrete cued-from-context cued conditioned responses (i.e., “the baseline issue”). The preclinical study of generalization is highly pertinent in the context of fear learning and memory because a lack of fear discrimination (overgeneralization) likely contributes to the etiology of anxiety-related disorders and post-traumatic stress disorder. © 2020 by John Wiley & Sons, Inc.Basic Protocol 1: Tone cued fear generalization gradientBasic Protocol 2: Quantification of freezingSupport Protocol: Alternate conditioning paradigms","PeriodicalId":40016,"journal":{"name":"Current Protocols in Neuroscience","volume":"91 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-01-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpns.89","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37590653","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 9

Investigating Memory Updating in Mice Using the Objects in Updated Locations Task 使用更新位置任务中的对象研究小鼠的记忆更新

Current Protocols in Neuroscience Pub Date : 2020-01-27 DOI: 10.1002/cpns.87

Destiny S. Wright, Kasuni K. Bodinayake, Janine L. Kwapis

{"title":"Investigating Memory Updating in Mice Using the Objects in Updated Locations Task","authors":"Destiny S. Wright, Kasuni K. Bodinayake, Janine L. Kwapis","doi":"10.1002/cpns.87","DOIUrl":"10.1002/cpns.87","url":null,"abstract":"In the laboratory, memory is typically studied as a de novo experience, in which a naïve animal is exposed to a discrete learning event that is markedly different from its past experiences. Most real-world memories, however, are updates—modifications or additions—to existing memories. This is particularly true in the aging, experienced brain. To better understand memory updating, we have developed a new behavioral paradigm called the objects in updated locations (OUL) task. OUL relies on hippocampus-dependent spatial learning and has the advantage of being able to test both the original memory and the updated information in a single test session. Further, OUL relies on incidental learning that avoids unnecessary stress that might hinder the performance of aging animals. In OUL, animals first learn the location of two identical objects in a familiar context. This memory is then updated by moving one object to a new location. Finally, to assess the animals’ memory for the original and the updated information, all animals are given a test session in which they are exposed to four copies of the object: two in the original training locations, one in the updated location, and one in a novel location. By comparing exploration of the novel location to the familiar locations, we can infer whether the animal remembers the original and updated object locations. OUL is a simple but powerful task that could provide new insights into the cellular, circuit-level, and molecular mechanisms that support memory updating. © 2020 by John Wiley & Sons, Inc.","PeriodicalId":40016,"journal":{"name":"Current Protocols in Neuroscience","volume":"91 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-01-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpns.87","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37581340","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 10

Overview of MARCM-Related Technologies in Drosophila Neurobiological Research 果蝇神经生物学研究中marcm相关技术综述

Current Protocols in Neuroscience Pub Date : 2020-01-23 DOI: 10.1002/cpns.90

Tsai-Chi Hsu, Kai-Yuan Ku, Hung-Chang Shen, Hung-Hsiang Yu

{"title":"Overview of MARCM-Related Technologies in Drosophila Neurobiological Research","authors":"Tsai-Chi Hsu, Kai-Yuan Ku, Hung-Chang Shen, Hung-Hsiang Yu","doi":"10.1002/cpns.90","DOIUrl":"10.1002/cpns.90","url":null,"abstract":"Mosaic analysis with a repressible cell marker (MARCM)–related technologies are positive genetic mosaic labeling systems that have been widely applied in studies of Drosophila brain development and neural circuit formation to identify diverse neuronal types, reconstruct neural lineages, and investigate the function of genes and molecules. Two types of MARCM-related technologies have been developed: single-colored and twin-colored. Single-colored MARCM technologies label one of two twin daughter cells in otherwise unmarked background tissues through site-specific recombination of homologous chromosomes during mitosis of progenitors. On the other hand, twin-colored genetic mosaic technologies label both twin daughter cells with two distinct colors, enabling the retrieval of useful information from both progenitor-derived cells and their subsequent clones. In this overview, we describe the principles and usage guidelines for MARCM-related technologies in order to help researchers employ these powerful genetic mosaic systems in their investigations of intricate neurobiological topics. © 2020 by John Wiley & Sons, Inc.","PeriodicalId":40016,"journal":{"name":"Current Protocols in Neuroscience","volume":"91 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpns.90","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37571452","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 2