Current Protocols in Neuroscience最新文献

Cannabinoid-induced tetrad is a preclinical model commonly used to evaluate if a pharmacological compound is an agonist of the central type-1 cannabinoid (CB1) receptor in rodents. The tetrad is characterized by hypolocomotion, hypothermia, catalepsy, and analgesia, four phenotypes that are induced by acute administration of CB1 agonists exemplified by the prototypic cannabinoid delta-9-tetrahydrocannabinol (THC). This unit describes a standard protocol in mice to induce tetrad phenotypes with THC as reference cannabinoid. We provide typical results obtained with this procedure showing a dose effect of THC in different mouse strains. The effect of the CB1 antagonist rimonabant is also shown. This tetrad protocol is well adapted to reveal new compounds acting on CB1 receptors in vivo. © 2017 by John Wiley & Sons, Inc.

In this unit, we describe in detail the most common methods used to break immunological tolerance for central myelin antigens and induce experimental autoimmune encephalomyelitis (EAE) in Lewis rats as an animal model of multiple sclerosis. The resulting disease course ranges from an acute monophasic disease to a chronic relapsing or chronic progressive course, which strongly resembles the human disease. These models enable the study of cellular and humoral autoimmunity against major antigenic epitopes of the myelin basic protein, myelin oligodendrocyte glycoprotein, or proteolipid protein. We provide an overview of common immunization protocols for induction of active and passive EAE, assessment and analysis of clinical score, preparation and purification of myelin basic protein, and derivation of neuroantigen-specific rat T cell lines. Finally, we describe the major clinical characteristics of these models. © 2017 by John Wiley & Sons, Inc.

Anatomical labeling approaches are essential for understanding brain organization. Among these approaches are various methods of performing tract tracing. However, a major hurdle to overcome when marking neurons in vivo is visibility. Poor visibility makes it challenging to image a desired neuronal pathway so that it can be easily differentiated from a closely neighboring pathway. As a result, it becomes impossible to analyze individual projections or their connections. The tracer that is chosen for a given purpose has a major influence on the quality of the tracing. Here, we describe the wheat germ agglutinin (WGA) tracer conjugated to Alexa fluorophores for reliable high-resolution tracing of central nervous system projections. Using the mouse cerebellum as a model system, we implement WGA-Alexa tracing for marking and mapping neural circuits that control motor function. We also show its utility for marking localized regions of the cerebellum after performing single-unit extracellular recordings in vivo. © 2017 by John Wiley & Sons, Inc.

Recording neural activity in awake, freely moving mice is a powerful and flexible technique for dissecting the neural circuit mechanisms underlying pathological behavior. This unit describes protocols for designing a drive and recording single neurons and local field potentials during anxiety-related paradigms. We also include protocols for integrating pharmacologic and optogenetic means for circuit manipulations, which, when combined with electrophysiological recordings, demonstrate input-specific and cell-specific contributions to circuit-wide activity. We discuss the planning, execution, and troubleshooting of physiology experiments during anxiety-like behavior. © 2017 by John Wiley & Sons, Inc.

The field of fluorescence microscopy is rapidly growing and offers ever more imaging capabilities for biologists. Over the past decade, many new technologies and techniques have been developed that allow for combinations of deeper, faster, and higher resolution imaging. These have included the commercialization of many super-resolution and light sheet fluorescence microscopy techniques. For the non-expert, it can be difficult to match the best imaging techniques to biological questions. Picking the most appropriate imaging modality requires a basic understanding of the underlying physics governing each of them, as well as information comparing potentially competing imaging properties in the context of the sample to be imaged. To address these issues, we provide here concise descriptions of a wide range of commercially available imaging techniques from wide-field to super-resolution microscopy, and provide a tabular guide to aid in comparisons among them. In this manner we provide a concise guide to understanding and matching the correct imaging modality to meet research needs. © 2017 by John Wiley & Sons, Inc.

Traumatic brain injury (TBI) is a major cause of death and disability world-wide. Following initial injury, TBI patients can face long-term disability in the form of cognitive, physical, and psychological deficits, depending on the severity and location of injury. This results in an economic burden in the United States estimated to be $60 billion due to health-care costs and loss of productivity. TBI is a significant area of active research interest for both military and civilian medicine. Numerous pre-clinical animal models of TBI are used to characterize the anatomical and physiological pathways involved and to evaluate therapeutic interventions. Due to its flexibility and scalability, controlled cortical impact (CCI) is one of the most commonly used preclinical TBI models. This unit provides a basic CCI protocol performed in the rat. © 2017 by John Wiley & Sons, Inc.

G protein–coupled receptors (GPCRs) comprise the single most targeted protein class in pharmacology. G protein signaling transduces extracellular stimuli such as neurotransmitters into cellular responses. Although preference for a specific GPCR among different G protein families (e.g., Gs-, Gi-, or Gq-like proteins) is often well studied, preference for a specific G protein subtype (e.g., Gi1, Gi2, Gi3, Go1, and Go2) has received little attention. Due to tissue expression differences and potentially exploitable functional differences, G protein subtype-dependent functional selectivity is an attractive framework to expand GPCR drug development. Herein we present a bioluminescence resonance energy transfer (BRET)-based method to characterize functional selectivity among Gi-like protein subtypes. © 2017 by John Wiley & Sons, Inc.

Nicotine, the main addictive component of tobacco, induces potentiation of brain stimulation reward, increases locomotor activity, and induces conditioned place preference. Nicotine cessation produces a withdrawal syndrome that can be relieved by nicotine replacement therapy. In the last decade, the market for electronic cigarettes has flourished, especially among adolescents. The nicotine vaporizer or electronic nicotine delivery system is a battery-operated device that allows the user to simulate the experience of tobacco smoking without inhaling smoke. The device is designed to be an alternative to conventional cigarettes that emits vaporized nicotine inhaled by the user. This report describes a procedure to vaporize nicotine in the air to produce blood nicotine levels in rodents that are clinically relevant to those that are observed in humans and produce dependence. We also describe how to construct the apparatus to deliver nicotine vapor in a stable, reliable, and consistent manner, as well as how to analyze air for nicotine content. © 2017 by John Wiley & Sons, Inc.

Autofluorescence is a problem that interferes with immunofluorescent staining and complicates data analysis. Throughout the mouse embryo, red blood cells naturally fluoresce across multiple wavelengths, spanning the emission and excitation spectra of many commonly used fluorescent reporters, including antibodies, dyes, stains, probes, and transgenic proteins, making it difficult to distinguish assay fluorescence from endogenous fluorescence. Several tissue treatment methods have been developed to bypass this issue with varying degrees of success. Sudan Black B dye has been commonly used to quench autofluorescence, but can also introduce background fluorescence. Here we present a protocol for an alternative called TrueBlack Lipofuscin Autofluorescence Quencher. The protocol described in this unit demonstrates how TrueBlack efficiently quenches red blood cell autofluorescence across red and green wavelengths in fixed embryonic tissue without interfering with immunofluorescent signal intensity or introducing background staining. We also identify optimal incubation, concentration, and multiple usage conditions for routine immunofluorescence microscopy. © 2017 by John Wiley & Sons, Inc.

This unit provides a step-by-step protocol for constructing cell type- and mitochondria-targeted GCaMP genetically encoded Ca²⁺ indicators (GECIs) for mitochondrial Ca²⁺ imaging in astrocytes. Mitochondrial Ca²⁺ plays a critical role in controlling cytosolic Ca²⁺ buffering, energy metabolism, and cellular signal transduction. Mitochondrial Ca²⁺ overload contributes to various pathological conditions, including neurodegeneration and apoptotic cell death in neurological diseases. Live-cell mitochondrial Ca²⁺ imaging is an important approach to understand mitochondrial Ca²⁺ dynamics and thus cell physiology and pathology. We implement astrocyte-specific mitochondrial targeting of GCaMP5G/6s (mito-GCaMP5G/6s). By loading X-Rhod-1 into astrocytes, we can simultaneously image mitochondrial and cytosolic Ca²⁺ signals. This protocol provides a novel approach to image mitochondrial Ca²⁺ dynamics as well as Ca²⁺ interplay between the endoplasmic reticulum and mitochondria. © 2018 by John Wiley & Sons, Inc.