Current Protocols in Neuroscience最新文献

Cover: In Erben and Buonanno (https://doi.org/10.1002/cpns63), the image shows examples for chromogenic BaseScope detection (Basic Protocol 2) and ISH-IHC combination (Basic Protocol 3). (A) Hippocampal section of an adult WT mice, labeled using BaseScope for ErbB4 with a single probe pair targeting the exon boundary exon 1/exon 2. The section was counterstained with hematoxylin and signal detected with light microscopy. Arrowheads indicate ErbB4+ GABAergic interneurons. (B) Detection of one of the four ErbB4 splice variants (JMb; cyan) by BaseScope in GABAergic interneurons (GAD-GFP; green; arrowheads) in a section of an adult GAD-GFP mouse (kindly provided by Dr. Yuchio Yanagawa). GFP signal was amplified after the ISH assay with an anti-GFP antibody (NeuroMab; N86/8; RRID: AB_10671444). (C) ISH for ErbB4 (C1; cyan) and Th (C3; magenta) was combined with IHC with an antibody against DAT (green; Santa Cruz, sc-32258; RRID: AB_627400) in a sagittal FFPE section from an adultWT mouse; depicted substantia nigra compacta (SNc) on the left and dopaminergic medial forebrain bundle. (C′) Magnification of ErbB4-expressing dopaminergic neurons (arrowheads) in the SNc. (D) In primary mesencephalic cultures (DIV8; prepared as in Skirzewski et al., 2018), DAT (green) immunostaining was performed post-hoc to RNAscope for ErbB4 (C1; cyan) and Th (C3; magenta). Scale bars 100 µm in B, 20 µm in other panels. Abbreviations: ISH-IHC, in situ hybridization-immunohistochemistry; GFP, green fluorescent protein; FFPE, formalin-fixed paraffin-embedded; WT, wild type.

Recombinant viruses are highly efficient vehicles for in vivo gene delivery. Viral vectors expand the neurobiology toolbox to include direct and rapid anterograde, retrograde, and trans-synaptic delivery of tracers, sensors, and actuators to the mammalian brain. Each viral type offers unique advantages and limitations. To establish strategies for selecting a suitable viral type, this article aims to provide readers with an overview of viral recombinant technology, viral structure, tropism, and differences between serotypes and pseudotypes for three of the most commonly used vectors in neurobiology research: adeno-associated viruses, retro/lentiviruses, and glycoprotein-deleted rabies viruses. © 2019 by John Wiley & Sons, Inc.

Advances in design and use of light-sensitive and light-emitting sensors have facilitated observation, measurement, and control of neuronal activities. Viruses are effective vectors for delivery of these valuable research tools to mammalian brains. Recombinant viruses are optimized to mediate regulatable, long-term, and cell-specific gene expression. Here, we describe production methods for three of the most commonly used types of recombinant viruses in neurobiology research: adeno-associated virus (AAV), retrovirus/lentivirus, and glycoprotein-deleted rabies virus. These viral constructs are frequently used for calcium imaging or to deliver neural tracers and optogenetic tools. Popular constructs are readily obtained commercially; however, customized virus production through commercial sources is time consuming and costly. This article aims to provide readers with detailed technical information for rapid production and validation of high-quality viral particles in a laboratory setting while highlighting advantages and limitations of each viral type. © 2019 by John Wiley & Sons, Inc.

Interneurons in the olfactory bulb are generated from neuronal precursor cells migrating from the anterior subventricular zone (SVZa) throughout the embryonic and postnatal life of mammals. This article describes basic methods for in vivo electroporation to label SVZa cells of both embryonic and postnatal rats. In addition, it describes three methods for tracing SVZa progenitors and following their migration pathway and differentiation, including immunohistochemistry, time-lapse live imaging in slice culture, and time-lapse imaging following transplantation in slice culture. These methods may be applied to all strains of rats and mice, including reporter mice. They may also be combined with methods such as BrdU labeling, tamoxifen injection, and electrophysiology, allowing one to observe proliferation or control gene expression at specific times and for specific neuronal functions. With time-lapse live imaging, details of labeled cells can be studied, including morphology, motility pattern, differentiation, and crosstalk between cells. © 2019 by John Wiley & Sons, Inc.

The calyx-type synapse is a giant synaptic structure in which a presynaptic terminal wraps around a postsynaptic neuron in a one-to-one manner. It has been used for decades as an experimental model system of the synapse due to its simplicity and high accessibility in physiological recording methods. In particular, the calyx of the embryonic chick ciliary ganglion (CG) has enormous potential for synapse science because more flexible genetic manipulations are available compared with other synapses. Here, we describe methods to study presynaptic morphology, physiology, and development using CGs and cutting-edge molecular tools. We outline step-by-step protocols for presynaptic gene manipulation using in ovo electroporation, preparation of isolated CGs, 3-D imaging for single-axon tracing in transparent CGs, electrophysiology of the presynaptic terminal, and an all-optical approach using optogenetic molecular reagents. These methods will facilitate studies of the synapse and neuronal circuits in the future. © 2019 by John Wiley & Sons, Inc.

Fluorescent detection of transcripts using RNAscope has quickly become a standard in situ hybridization (ISH) approach in neuroscience with over 400 publications since its introduction in 2012. RNAscope's sensitivity and specificity allow the simultaneously detection of up to three low abundance mRNAs in single cells (i.e., multiplexing) and, in contrast to other ISH techniques, RNAscope is performed in 1 day. BaseScope, a newer ultrasensitive platform, uses improved amplification chemistry of single oligonucleotide probe pairs (∼50 bases). This technique allows discrimination of single nucleotide polymorphisms or splice variants that differ by short exons. A present limitation of BaseScope is that expression analysis is limited to a single gene (i.e., single-plexing). This article outlines detailed protocols for both RNAscope and BaseScope in neuronal tissue. We discuss how to perform ISH experiments using either fresh-frozen or formalin-fixed paraffin-embedded sections, as well as dissociated cultured neurons. We also outline how to obtain quantitative data from hybridized tissue sections. © 2019 by John Wiley & Sons, Inc.

In this unit, we describe an inexpensive and versatile method for optogenetic stimulation of a large population of genetically engineered Caenorhabditis elegans worms while quantitatively analyzing behavior. A custom light-emitting diode light source is used to deliver blue-light stimuli, causing direct depolarization of neurons expressing the light-gated cation channel Channelrhodopsin-2, which in turn evokes behavioral responses. The behavioral responses are recorded by a high-throughput machine vision–based tracking system, the Multi-Worm Tracker, for detailed analysis. This approach allows researchers to bypass technical obstacles to simultaneously deliver uniform stimuli to a large number of freely behaving animals and investigate the neural underpinnings of behavior. © 2018 by John Wiley & Sons, Inc.

Visualizing neural activity from deep brain regions in freely behaving animals through miniature fluorescent microscope (miniscope) systems is becoming more important for understanding neural encoding mechanisms underlying cognitive functions. Here we present our custom-designed miniscope GRadient INdex (GRIN) lens system that enables simultaneously recording from hundreds of neurons for months. This article includes miniscope design, the surgical procedure for GRIN lens implantation, miniscope mounting on the head of a mouse, and data acquisition and analysis. First, a target brain region is labeled with virus expressing GCaMP6; second, a GRIN lens is implanted above the target brain region; third, following mouse surgical recovery, a miniscope is mounted on the head of the mouse above the GRIN lens; and finally, neural activity is recorded from the freely behaving mouse. This system can be applied to recording the same population of neurons longitudinally, enabling the elucidation of neural mechanisms underlying behavioral control. © 2018 by John Wiley & Sons, Inc.