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Current Protocols in Neuroscience

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https://www.readbyqxmd.com/read/29064566/generation-and-characterization-of-functional-human-hypothalamic-neurons
#1
Peter Kirwan, Magdalena Jura, Florian T Merkle
Neurons in the hypothalamus orchestrate homeostatic physiological processes and behaviors essential for life. Defects in the function of hypothalamic neurons cause a spectrum of human diseases, including obesity, infertility, growth defects, sleep disorders, social disorders, and stress disorders. These diseases have been studied in animal models such as mice, but the rarity and relative inaccessibility of mouse hypothalamic neurons and species-specific differences between mice and humans highlight the need for human cellular models of hypothalamic diseases...
October 24, 2017: Current Protocols in Neuroscience
https://www.readbyqxmd.com/read/29058772/controlled-cortical-impact-in-the-rat
#2
Dana D Dean, Joseph A Frank, L Christine Turtzo
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...
October 23, 2017: Current Protocols in Neuroscience
https://www.readbyqxmd.com/read/29058771/bioluminescence-resonance-energy-transfer-assay-to-characterize-gi-like-g-protein-subtype-dependent-functional-selectivity
#3
Hideaki Yano, Marta Sánchez-Soto, Sergi Ferré
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...
October 23, 2017: Current Protocols in Neuroscience
https://www.readbyqxmd.com/read/29058770/suppression-of-red-blood-cell-autofluorescence-for-immunocytochemistry-on-fixed-embryonic-mouse-tissue
#4
Niteace C Whittington, Susan Wray
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...
October 23, 2017: Current Protocols in Neuroscience
https://www.readbyqxmd.com/read/29058769/lewis-rat-model-of-experimental-autoimmune-encephalomyelitis
#5
Kalliopi Pitarokoili, Bjoern Ambrosius, Ralf Gold
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...
October 23, 2017: Current Protocols in Neuroscience
https://www.readbyqxmd.com/read/28678400/nicotine-vapor-method-to-induce-nicotine-dependence-in-rodents
#6
Marsida Kallupi, Olivier George
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...
July 5, 2017: Current Protocols in Neuroscience
https://www.readbyqxmd.com/read/28678399/a-guide-to-creating-and-testing-new-intrsect-constructs
#7
Lief E Fenno, Joanna Mattis, Charu Ramakrishnan, Karl Deisseroth
As the power of genetically encoded interventional and observational tools for neuroscience expands, the boundaries of experimental design are increasingly defined by limits in selectively expressing these tools in relevant cell types. Single-recombinase-dependent expression systems have been widely used as a means to restrict gene expression based on single features by combining recombinase-dependent viruses with recombinase-expressing transgenic animals. This protocol details how to create INTRSECT constructs and use multiple recombinases to achieve targeting of a desired gene to subsets of neurons that are defined by multiple genetic and/or topological features...
July 5, 2017: Current Protocols in Neuroscience
https://www.readbyqxmd.com/read/28678398/cannabinoid-induced-tetrad-in-mice
#8
Mathilde Metna-Laurent, Miguel Mondésir, Agnès Grel, Monique Vallée, Pier-Vincenzo Piazza
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...
July 5, 2017: Current Protocols in Neuroscience
https://www.readbyqxmd.com/read/28678397/multisite-electrophysiology-recordings-in-mice-to-study-cross-regional-communication-during-anxiety
#9
Alexander Z Harris, Danielle Golder, Ekaterina Likhtik
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...
July 5, 2017: Current Protocols in Neuroscience
https://www.readbyqxmd.com/read/28678396/animal-model-of-chronic-migraine-associated-pain
#10
Laura S Moye, Amynah A A Pradhan
Migraine is a debilitating condition that affects hundreds of millions of people worldwide. A subset of these patients experience chronic migraine, resulting in long-term disability and a severely lowered quality of life. The development of novel migraine therapies has been slow, partially due to the small number of predictive animal models. We have recently developed a novel model of chronic migraine-associated pain, using the known human migraine trigger, nitroglycerin. Injection of nitroglycerin evokes an acute mechanical hyperalgesia, which is sensitive to the acute migraine therapy sumatriptan...
July 5, 2017: Current Protocols in Neuroscience
https://www.readbyqxmd.com/read/28398646/fluorescein-isothiocyanate-fitc-dextran-extravasation-as-a-measure-of-blood-brain-barrier-permeability
#11
Reka Natarajan, Nicole Northrop, Bryan Yamamoto
The blood-brain barrier (BBB) is formed in part by vascular endothelial cells that constitute the capillaries and microvessels of the brain. The function of this barrier is to maintain homeostasis within the brain microenvironment and buffer the brain from changes in the periphery. A dysfunction of the BBB would permit circulating molecules and pathogens typically restricted to the periphery to enter the brain and interfere with normal brain function. As increased permeability of the BBB is associated with several neuropathologies, it is important to have a reliable and sensitive method that determines BBB permeability and the degree of BBB disruption...
April 10, 2017: Current Protocols in Neuroscience
https://www.readbyqxmd.com/read/28398645/crispr-cas9-mediated-gene-knockout-in-the-mouse-brain-using-in-utero-electroporation
#12
Yohei Shinmyo, Hiroshi Kawasaki
This unit describes a highly efficient and rapid procedure for brain-specific disruption of genes in the developing mouse brain using pX330 plasmids expressing humanized Cas9 and single-guide RNAs (sgRNAs) against target genes. The pX330 plasmids are delivered into the rodent brain using in utero electroporation. Focusing on the Satb2 gene, which encodes an AT-rich DNA-binding transcription factor, we found that the introduction of pX330-Satb2 induced insertion/deletion (indel) mutations near the predicted cleavage site in the Satb2 gene, resulting in a dramatic reduction of Satb2 expression in post-mitotic neurons...
April 10, 2017: Current Protocols in Neuroscience
https://www.readbyqxmd.com/read/28398644/quantitative-high-throughput-screening-using-a-coincidence-reporter-biocircuit
#13
Brittany W Schuck, Ryan MacArthur, James Inglese
Reporter-biased artifacts-i.e., compounds that interact directly with the reporter enzyme used in a high-throughput screening (HTS) assay and not the biological process or pharmacology being interrogated-are now widely recognized to reduce the efficiency and quality of HTS used for chemical probe and therapeutic development. Furthermore, narrow or single-concentration HTS perpetuates false negatives during primary screening campaigns. Titration-based HTS, or quantitative HTS (qHTS), and coincidence reporter technology can be employed to reduce false negatives and false positives, respectively, thereby increasing the quality and efficiency of primary screening efforts, where the number of compounds investigated can range from tens of thousands to millions...
April 10, 2017: Current Protocols in Neuroscience
https://www.readbyqxmd.com/read/28398643/systems-genetic-analysis-in-genenetwork-org
#14
Clarissa C Parker, Price E Dickson, Vivek M Philip, Mary Thomas, Elissa J Chesler
Genome-wide association studies (GWAS) have emerged as a powerful tool to identify alleles and molecular pathways that influence susceptibility to psychiatric disorders and other diseases. Forward genetics using mouse mapping populations allows for a complementary approach that provides rigorous genetic and environmental control. In this unit, we describe techniques and tools that reduce the technical burden traditionally associated with genetic mapping in mice and enhance their translational utility to human psychiatric disorders...
April 10, 2017: Current Protocols in Neuroscience
https://www.readbyqxmd.com/read/28398642/wga-alexa-conjugates-for-axonal-tracing
#15
Sabrina L Levy, Joshua J White, Elizabeth P Lackey, Lindsey Schwartz, Roy V Sillitoe
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...
April 10, 2017: Current Protocols in Neuroscience
https://www.readbyqxmd.com/read/28398641/time-lapse-imaging-and-cell-tracking-of-migrating-cells-in-slices-and-flattened-telencephalic-vesicles
#16
Verónica Murcia-Belmonte, Giovanna Expósito, Eloísa Herrera
Neuronal migration is a vital process needed for subsequent assembly and function of neural circuitry during embryonic development. The vast majority of neural progenitors are generated far from their final destination and need to migrate considerable distances to reach their specific cortical layer. Innovations in cell culture techniques and fluorescence microscopy now facilitate the direct visualization of cell movements during cortical development. Here, a detailed protocol to record and analyze a particular type of early migrating neurons, the Cajal Retzius Cells, during the development of the telencephalic vesicles in mammals is described...
April 10, 2017: Current Protocols in Neuroscience
https://www.readbyqxmd.com/read/28398640/fluorescence-microscopy-a-concise-guide-to-current-imaging-methods
#17
Christian A Combs, Hari Shroff
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...
April 10, 2017: Current Protocols in Neuroscience
https://www.readbyqxmd.com/read/28046204/electroporation-in-the-rodent-embryonic-brain-using-whole-embryo-culture-system
#18
Takako Kikkawa, Masanori Takahashi, Noriko Osumi
This unit describes basic methods for mammalian whole embryo culture (WEC) using embryonic day 10.5 mouse embryos, including the preparation of high-quality immediately centrifuged (IC) rat serum that is commonly used for WEC and is essential for normal growth and development of cultured mouse and rat embryos in vitro. An alternative protocol for different stages of rodent embryos is also introduced. Since embryos for WEC are dissected out of the uterus and manipulated under the microscope, one can overcome many of the difficulties of gene delivery encountered using in utero electroporation...
January 3, 2017: Current Protocols in Neuroscience
https://www.readbyqxmd.com/read/28046203/visualizing-changes-in-neuronal-dendritic-morphology-in-response-to-stress-and-pharmacological-challenge
#19
Cara L Wellman
This unit outlines a protocol for Golgi staining, which has been used extensively to reliably and quantitatively assess alterations in dendritic arborization and spine density as a result of a variety of factors, including chronic administration of glucocorticoids, chronic stress, and pharmacological manipulations. The method stains neurons in their entirety, allowing for sophisticated analyses of branch lengths and numbers as well as patterns of dendritic branching. Advantages of the technique include its usefulness in multisite collaborations and its utility in visualizing neurons in multiple regions within the same brain...
January 3, 2017: Current Protocols in Neuroscience
https://www.readbyqxmd.com/read/28046202/intracerebral-injections-and-ultrastructural-analysis-of-high-pressure-frozen-brain-tissue
#20
Marie-Theres Weil, Torben Ruhwedel, Wiebke Möbius, Mikael Simons
Intracerebral injections are an invasive method to bypass the blood brain barrier and are widely used to study molecular and cellular mechanisms of the central nervous system. The administered substances are injected directly at the site of interest, executing their effect locally. By combining injections in the rat brain with state-of-the-art electron microscopy, subtle changes in ultrastructure of the nervous tissue can be detected prior to overt damage or disease. The protocol presented here involves stereotactic injection into the corpus callosum of Lewis rats and the cryopreparation of freshly dissected tissue for electron microscopy...
January 3, 2017: Current Protocols in Neuroscience
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