Attentional set shifting is a measure of cognitive flexibility and executive functions widely assessed in humans by the Wisconsin Card Sorting Test (WCST) and the CANTAB Intra-/Extra-Dimensional set-shifting task (ID/ED). The recently established automated two-chamber "Operon ID/ED" task for mice has proved to be an effective preclinical tool for drug testing and genetic screening, with direct translational valence in healthy human subjects and patients with schizophrenia. Here, we describe an upgraded version of the Operon ID/ED task that is now commercially available. This automated task allows one to study the ability of mice to shift attention through different rules, using two or three different dimensions (i.e., lights, odors, and textures). This unit provides a detailed step-by-step protocol for preparing and testing the mice that includes all procedures required for this upgraded attentional set-shifting paradigm. A short manual for the use of the dedicated ANY-maze software and tools for adapting it to different needs are also provided. Overall, this is a comprehensive guideline for the use of this complex upgraded equipment and paradigm. © 2020 Wiley Periodicals LLC. Basic Protocol: Operon ID/ED testing Support Protocol: Use of ANY-maze software.

Astrocytes are actively involved in a neuroprotective role in the brain, which includes scavenging reactive oxygen species to minimize tissue damage. They also modulate neuroinflammation and reactive gliosis prevalent in several brain disorders like epilepsy, Alzheimer's, and Parkinson's disease. In animal models, targeted manipulation of astrocytic function via modulation of their calcium (Ca2+ ) oscillations by incorporating light-sensitive cation channels like Channelrhodopsin-2 (ChR2) offers a promising avenue in influencing the long-term progression of these disorders. However, using adult animals for Ca2+ imaging poses major challenges, including accelerated deterioration of in situ slice health and age- related changes. Additionally, optogenetic preparations necessitate usage of a red-shifted Ca2+ indicator like Rhod-2 AM to avoid overlapping light issues between ChR2 and the Ca2+ indicator during simultaneous optogenetic stimulation and imaging. In this article, we provide an experimental setting that uses live adult murine brain slices (2-5 months) from a knock-in model expressing Channelrhodopsin-2 (ChR2(C128S)) in cortical astrocytes, loaded with Rhod-2 AM to elicit robust Ca2+ response to light stimulation. We have developed and standardized a protocol for brain extraction, sectioning, Rhod-2 AM loading, maintenance of slice health, and Ca2+ imaging during light stimulation. This has been successfully applied to optogenetically control adult cortical astrocytes, which exhibit synchronous patterns of Ca2+ activity upon light stimulation, drastically different from resting spontaneous activity. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Experimental preparation, setup, slice preparation and Rhod-2 AM staining Basic Protocol 2: Image acquisition and analysis.

Fluorescence lifetime microscopy (FLIM) and Förster's resonance energy transfer (FRET) are advanced optical tools that neuroscientists can employ to interrogate the structure and function of complex biological systems in vitro and in vivo using light. In neurobiology they are primarily used to study protein-protein interactions, to study conformational changes in protein complexes, and to monitor genetically encoded FRET-based biosensors. These methods are ideally suited to optically monitor changes in neurons that are triggered optogenetically. Utilization of this technique by neuroscientists has been limited, since a broad understanding of FLIM and FRET requires familiarity with the interactions of light and matter on a quantum mechanical level, and because the ultra-fast instrumentation used to measure fluorescent lifetimes and resonance energy transfer are more at home in a physics lab than in a biology lab. In this overview, we aim to help neuroscientists overcome these obstacles and thus feel more comfortable with the FLIM-FRET method. Our goal is to aid researchers in the neuroscience community to achieve a better understanding of the fundamentals of FLIM-FRET and encourage them to fully leverage its powerful ability as a research tool. Published 2020. U.S. Government.

Nanobodies (nAbs) are recombinant antigen-binding variable domain fragments obtained from heavy-chain-only immunoglobulins. Among mammals, these are unique to camelids (camels, llamas, alpacas, etc.). Nanobodies are of great use in biomedical research due to their efficient folding and stability under a variety of conditions, as well as their small size. The latter characteristic is particularly important for nAbs used as immunolabeling reagents, since this can improve penetration of cell and tissue samples compared to conventional antibodies, and also reduce the gap distance between signal and target, thereby improving imaging resolution. In addition, their recombinant nature allows for unambiguous definition and permanent archiving in the form of DNA sequence, enhanced distribution in the form of sequences or plasmids, and easy and inexpensive production using well-established bacterial expression systems, such as the IPTG induction method described here. This article will review the basic workflow and process for developing, screening, and validating novel nAbs against neuronal target proteins. The protocols described make use of the most common nAb development method, wherein an immune repertoire from an immunized llama is screened via phage display technology. Selected nAbs can then be taken through validation assays for use as immunolabels or as intrabodies in neurons. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Total RNA isolation from camelid leukocytes Basic Protocol 2: First-strand cDNA synthesis; VH H and VH repertoire PCR Basic Protocol 3: Preparation of the phage display library Basic Protocol 4: Panning of the phage display library Basic Protocol 5: Small-scale nAb expression Basic Protocol 6: Sequence analysis of selected nAb clones Basic Protocol 7: Nanobody validation as immunolabels Basic Protocol 8: Generation of nAb-pEGFP mammalian expression constructs Basic Protocol 9: Nanobody validation as intrabodies Support Protocol 1: ELISA for llama serum testing, phage titer, and screening of selected clones Support Protocol 2: Amplification of helper phage stock Support Protocol 3: nAb expression in amber suppressor E. coli bacterial strains.

Basic neuroscience research employs antibodies as key reagents to label, capture, and modulate the function of proteins of interest. Antibodies are immunoglobulin proteins. Recombinant antibodies are immunoglobulin proteins whose nucleic acid coding regions, or fragments thereof, have been cloned into expression plasmids that allow for unlimited production. Recombinant antibodies offer many advantages over conventional antibodies including their unambiguous identification and digital archiving via DNA sequencing, reliable expression, ease and reliable distribution as DNA sequences and as plasmids, and the opportunity for numerous forms of engineering to enhance their utility. Recombinant antibodies exist in many different forms, each of which offers potential advantages and disadvantages for neuroscience research applications. I provide an overview of recombinant antibodies and their development. Examples of their emerging use as valuable reagents in basic neuroscience research are also discussed. Many of these examples employ recombinant antibodies in innovative experimental approaches that cannot be pursued with conventional antibodies. © 2020 Wiley Periodicals LLC.

Neuronal mitochondrial fragmentation is a phenotype exhibited in models of neurodegeneration such as Parkinson's disease. Delineating the dysfunction in mitochondrial dynamics found in diseased states can aid our understanding of underlying mechanisms of disease progression and possibly identify novel therapeutic approaches. Advances in microscopy and the availability of intuitive open-access software have accelerated the rate of image acquisition and analysis, respectively. These developments allow routine biology researchers to rapidly turn hypotheses into results. In this protocol, we describe the utilization of cell culture techniques, high-content imaging (HCI), and the subsequent open-source image analysis pipeline for the quantification of mitochondrial fragmentation in the context of a rotenone-based in vitro Parkinson's disease model. © 2020 The Authors. Basic Protocol 1: SN4741 neuron culture and treatment in a rotenone-based model of Parkinson's disease Basic Protocol 2: Identification of cell nuclei, measurement of mitochondrial membrane potential, and measurement of mitochondrial fragmentation in mouse-derived midbrain dopaminergic neurons.

MagellanMapper is a software suite designed for visual inspection and end-to-end automated processing of large-volume, 3D brain imaging datasets in a memory-efficient manner. The rapidly growing number of large-volume, high-resolution datasets necessitates visualization of raw data at both macro- and microscopic levels to assess the quality of data, as well as automated processing to quantify data in an unbiased manner for comparison across a large number of samples. To facilitate these analyses, MagellanMapper provides both a graphical user interface for manual inspection and a command-line interface for automated image processing. At the macroscopic level, the graphical interface allows researchers to view full volumetric images simultaneously in each dimension and to annotate anatomical label placements. At the microscopic level, researchers can inspect regions of interest at high resolution to build ground truth data of cellular locations such as nuclei positions. Using the command-line interface, researchers can automate cell detection across volumetric images, refine anatomical atlas labels to fit underlying histology, register these atlases to sample images, and perform statistical analyses by anatomical region. MagellanMapper leverages established open-source computer vision libraries and is itself open source and freely available for download and extension. © 2020 Wiley Periodicals LLC. Basic Protocol 1: MagellanMapper installation Alternate Protocol: Alternative methods for MagellanMapper installation Basic Protocol 2: Import image files into MagellanMapper Basic Protocol 3: Region of interest visualization and annotation Basic Protocol 4: Explore an atlas along all three dimensions and register to a sample brain Basic Protocol 5: Automated 3D anatomical atlas construction Basic Protocol 6: Whole-tissue cell detection and quantification by anatomical label Support Protocol: Import a tiled microscopy image in proprietary format into MagellanMapper.