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Current Protocols in Neuroscience

Emily E Kramer, Patrick E Steadman, Jonathan R Epp, Paul W Frankland, Sheena A Josselyn
Arc (activity-regulated cytoskeleton-associated protein) is an immediate early gene that may be used to label recently active neurons. Arc is transcribed following neuronal activity, and its mRNA is then rapidly transported to dendrites. This feature allows nuclear-localized Arc mRNA to define ensembles of recently active neurons in systems or circuit neuroscience. However, typical in situ hybridization techniques severely constrain the thickness of the tissue specimen (typically 20-µm brain slices). Here, we describe a protocol for visualizing intranuclear Arc mRNA in large (4 × 4 × 3 mm) volumes of intact mouse brain tissue...
June 26, 2018: Current Protocols in Neuroscience
Alexander D Jacob, Adam I Ramsaran, Andrew J Mocle, Lina M Tran, Chen Yan, Paul W Frankland, Sheena A Josselyn
Miniaturized fluorescence microscopes for imaging calcium transients are a promising tool for investigating the relationship between behavior and population-level neuronal activity in rodents. However, commercially available miniature microscopes may be costly and, because they are closed source, may not be easily modified based on particular experimental requirements. Here, we describe how to build and use a low-cost compact head-mounted endoscope (CHEndoscope) system for in vivo calcium imaging. The CHEndoscope uses an implanted gradient index lens along with the genetically encoded calcium indicator GCaMP6 to image calcium transients from hundreds of neurons simultaneously in awake behaving mice...
June 26, 2018: Current Protocols in Neuroscience
Alexei Morozov
The lasting behavioral changes elicited by social signals provide important adaptations for survival of organisms that thrive as a group. Unlike the rapid innate responses to social cues, such adaptations have been understudied. Here, the rodent models of the lasting socially induced behavioral changes are presented as either modulations or reinforcements of the distinct forms of learning and memory or non-associative changes of affective state. The purpose of this categorization is to draw attention to the potential mechanistic links between the neuronal pathways that process social cues and the neuronal systems that mediate the well-studied forms of learning and memory...
May 16, 2018: Current Protocols in Neuroscience
Nannan Zhang, Shinghua Ding
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...
January 22, 2018: Current Protocols in Neuroscience
W Scott Young, June Song, Éva Mezey
This unit presents protocols to locate RNA transcripts in tissues. Numerous approaches are detailed, including those that use radiolabeled or colorimetric probes. Also, the probes may be modified oligodeoxynucleotides, singly or in pairs, as well as ribonucleic acids. High sensitivity and specificity are obtained, especially with sets of oligodeoxynucleotide pairs. © 2018 by John Wiley & Sons, Inc.
January 22, 2018: Current Protocols in Neuroscience
Rand R Wilcox, Guillaume A Rousselet
There is a vast array of new and improved methods for comparing groups and studying associations that offer the potential for substantially increasing power, providing improved control over the probability of a Type I error, and yielding a deeper and more nuanced understanding of data. These new techniques effectively deal with four insights into when and why conventional methods can be unsatisfactory. But for the non-statistician, the vast array of new and improved techniques for comparing groups and studying associations can seem daunting, simply because there are so many new methods that are now available...
January 22, 2018: Current Protocols in Neuroscience
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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