Current Protocols in Neuroscience最新文献

Recombinant adeno-associated virus (rAAV) is a mammalian virus that has been altered to be used as a gene delivery vehicle. Several changes to the viral genome have made them replication deficient so that this aspect of the viral infection cycle is under full control of the experimenter, while maintaining gene expression machinery. Over the last decades, rAAVs have become the gold standard for studying in vivo gene function and are especially favorable for gene transfer in the central nervous system. AAVs have been proven safe and provide stable gene expression over a long period of time. They are extensively used in preclinical experiments and show great potential for clinical applications. However, the use of AAVs in preclinical settings are often held back due to availability. Waiting lines are long at commercial production facilities, and in-lab production is hindered due to lack of specific laboratory equipment needed. Here we present a novel production method that can be carried out in any molecular biology laboratory using standard laboratory equipment. We provide a simple, fast, and streamlined protocol for production that can result in titers comparable with the more time-consuming iodixanol gradient ultracentrifugation method. The yield using this protocol is high enough for any type of study where AAV is the vector of choice. © 2020 The Authors.

Elucidating the complex neural mechanisms that underlie cognition is contingent upon our ability to measure behavioral outputs reliably in animal models. While the development of open-source software has made behavioral science more accessible, behavioral research remains underappreciated and underutilized. One reason is the large real estate necessitated by traditional behavioral setups. Space must be specifically allocated for a controlled testing environment, accommodate the large footprint of mazes used in behavioral research, and allow a contiguous computerized area for data acquisition. Additionally, to achieve the distinct and sometimes incompatible environmental conditions required by different tasks, a suite of testing rooms may be necessary. Because space is a limited resource, this makes behavioral testing impractical for some labs or leads to implementation of suboptimal solutions that compromise the ergonomics of the working space, prevent the adequate control of environmental parameters around the testing setup, and jeopardize experimental reproducibility. Here, we describe a modular, space-saving, self-sufficient, functional, customizable, and cost-efficient setup to allow a large line of behavioral tests in mice within a single, compact room (<8 m² ). Because it is modular by design, this setup requires no compromises on ergonomics, environmental control, or complexity of the visual landscape. It is inherently effective at streamlining behavioral experiments by eliminating the need to redefine tracking parameters, and makes swapping between configurations fast (∼1 min) and effortless. Presently, this design allows one to run eight major behavioral tasks, permitting a detailed and comprehensive analysis of mouse behavior within the footprint of a small office. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Constructing the support table Support Protocol 1: Constructing the open-field maze Support Protocol 2: Constructing IR-permissive inserts for light-dark assays Support Protocol 3: Constructing the three-chamber maze Support Protocol 4: Constructing the Y maze Support Protocol 5: Constructing the elevated plus maze Support Protocol 6: Constructing the Barnes maze Basic Protocol 2: Setting up the behavior room: flange and pulley systems Basic Protocol 3: Setting up the behavior room: environmental and storage systems Basic Protocol 4: Assembling and switching between configurations.

Expansion microscopy (ExM) is a technique that physically expands preserved cells and tissues before microscope imaging, so that conventional diffraction-limited microscopes can perform nanoscale-resolution imaging. In ExM, biomolecules or their markers are linked to a dense, swellable gel network synthesized throughout a specimen. Mechanical homogenization of the sample (e.g., by protease digestion) and the addition of water enable isotropic swelling of the gel, so that the relative positions of biomolecules are preserved. We previously presented ExM protocols for analyzing proteins and RNAs in cells and tissues. Here we describe a cookbook-style ExM protocol for expanding cultured HeLa cells with immunostained microtubules, aimed to help newcomers familiarize themselves with the experimental setups and skills required to successfully perform ExM. Our aim is to help beginners, or students in a wet-lab classroom setting, learn all the key steps of ExM. © 2020 The Authors.

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAARs), exhibit highly dynamic trafficking and cell surface mobility. Regulated trafficking to and from the surface is a critical determinant of GABAAR neurotransmission. Receptors delivered by exocytosis diffuse laterally in the plasma membrane, with tethering and reduced movement at synapses occurring through receptor interactions with the subsynaptic scaffold. After diffusion away from synapses, receptors are internalized by clathrin-dependent endocytosis at extrasynaptic sites and can be either recycled back to the cell membrane or degraded in lysosomes. To study the dynamics of these key trafficking events in neurons, we have developed novel optical methods based around receptors containing a dual-tagged γ2 subunit (γ2pHFAP) in combination with fluorogen dyes. Specifically, the GABAAR γ2 subunit is tagged with a pH-sensitive green fluorescent protein and a fluorogen-activating peptide (FAP). The FAP allows receptor labeling with fluorogen dyes that are optically silent until bound to the FAP. Combining FAP and fluorescent imaging with organelle labeling allows novel and accurate measurement of receptor turnover and accumulation into intracellular compartments under basal conditions in scenarios ranging from in vitro seizure models to drug exposure paradigms. Here we provide a protocol to track and quantify receptors in transit from the neuronal surface to endosomes and lysosomes. This protocol is readily applicable to cell lines and primary cells, allowing rapid quantitative measurements of receptor surface levels and postendocytic trafficking decisions. © 2020 by John Wiley & Sons, Inc.

Basic Protocol 1: Preparation of cortical neuronal cultures for imaging assays

Basic Protocol 2: Surface receptor internalization and trafficking to early endosomes

Basic Protocol 3: Measurement of receptor steady state surface level, synaptic level, and lysosomal targeting

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 apparatus

Basic Protocol 2: Head fixture surgery

Basic Protocol 3: General operation of the experimental apparatus

Basic Protocol 4: Behavioral task design and training

Basic Protocol 5: Psychometric data collection and analysis

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 slices

Basic Protocol 2: Preparation of gene gun bullets

Basic Protocol 3: Gene gun transfection of slices

Basic Protocol 4: Visualization and tracking of mitochondrial movement

Alternate Protocol: Transduction of EGFP-mito via viral injection of the neonatal mouse brain

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 homozygosity

Support Protocol 1: Detection of ectopic Cre recombinase expression

Basic Protocol 2: The RiboTag assay

Support Protocol 2: Real-time quantitative PCR (qRT-PCR) assay of RiboTag-derived cell-type-specific RNA

Support Protocol 3: Construction of cell-type-specific RNA-seq library

Support Protocol 4: Secondary analyses of RiboTag-derived RNA-seq dataset

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.

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.

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.