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Current Protocols in Neuroscience

Alex J Yu, Troy A McDiarmid, Evan L Ardiel, Catharine H Rankin
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...
November 2, 2018: Current Protocols in Neuroscience
Lifeng Zhang, Bo Liang, Giovanni Barbera, Sarah Hawes, Yan Zhang, Kyle Stump, Ira Baum, Yupeng Yang, Yun Li, Da-Ting Lin
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...
October 13, 2018: Current Protocols in Neuroscience
Koen Kole, Tansu Celikel
The heterogeneous organization of the mammalian neocortex poses a challenge for elucidating the molecular mechanisms underlying its physiological processes. Although high-throughput molecular methods are increasingly deployed in neuroscience, their anatomical specificity is often lacking. In this unit, we introduce a targeted microdissection technique that enables extraction of high-quality RNA and proteins at high anatomical resolution from acutely prepared brain slices. We exemplify its utility by isolating single cortical columns and laminae from the mouse primary somatosensory (barrel) cortex...
October 4, 2018: Current Protocols in Neuroscience
Amanda R Pahng, Scott Edwards
In contrast to their analgesic properties, excessive use of either opioids or alcohol produces a paradoxical emergence of heightened pain sensitivity to noxious stimuli, termed hyperalgesia, which may promote increased use of opioids or alcohol drinking to manage worsening pain symptoms. Hyperalgesia has traditionally been measured in rodents via reflex-based assays, including the von Frey method. To better model the motivational and affective dimensions of pain in a state of opioid/alcohol dependence and withdrawal, this unit describes the use of a non-reflex-based method for measuring pain avoidance-like behavior in dependent rats...
October 2018: Current Protocols in Neuroscience
Varun Sagi, Waogwende L Song-Naba, Barbara A Benson, Sonal S Joshi, Kalpna Gupta
Sickle cell disease (SCD) is a genetic blood disorder that impacts millions of individuals worldwide. SCD is characterized by debilitating pain that can begin during infancy and may continue to increase throughout life. This pain can be both acute and chronic. A characteristic feature specific to acute pain in SCD occurs during vaso-occlusive crisis (VOC) due to the blockade of capillaries with sickle red blood cells. The acute pain of VOC is intense, unpredictable, and requires hospitalization. Chronic pain occurs in a significant population with SCD...
September 28, 2018: Current Protocols in Neuroscience
Bryan M Hooks
Manipulation of defined neurons using excitatory opsins, including channelrhodopsin, enables studies of connectivity and the functional role of these circuit components in the brain. These techniques are vital in the neocortex, where diverse neurons are intermingled, and stimulation of specific cell types is difficult without the spatial, temporal, and genetic control available with optogenetic approaches. Channelrhodopsins are effective for mapping excitatory connectivity from one input type to its target...
September 11, 2018: 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...
July 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...
July 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...
July 2018: Current Protocols in Neuroscience
Mayuko Kano, Hidetaka Suga, Takatoshi Kasai, Chikafumi Ozone, Hiroshi Arima
The anterior pituitary gland produces several hormones essential for regulation of the somatic endocrine system. Deficiency of these hormones can cause life-threatening diseases, including adrenal crisis. Pituitary tissue generated from human pluripotent stem cells is expected to provide better treatment than current hormone replacement therapy. During early mammalian development, the pituitary anlage (Rathke's pouch) develops from non-neural ectoderm adjacent to the developing ventral hypothalamus. The close interaction between these two tissues is crucial for Rathke's pouch development and involves several signaling molecules...
April 2018: Current Protocols in Neuroscience
Claudia Rangel-Barajas, George V Rebec
Transgenic mouse models of Huntington's disease (HD), a neurodegenerative condition caused by a single gene mutation, have been transformative in their ability to reveal the molecular processes and pathophysiological mechanisms underlying the HD behavioral phenotype. Three model categories have been generated depending on the genetic context in which the mutation is expressed: truncated, full-length, and knock-in. No single model, however, broadly replicates the behavioral symptoms and massive neuronal loss that occur in human patients...
April 2018: Current Protocols in Neuroscience
Tingting Yu, Dan Zhu
Tissue optical clearing techniques have provided essential tools for visualization of neural networks in unsectioned brain tissue. Here, we describe a passive optical clearing method based on hydrogel embedding, PACT (passive clarity technique), which is relatively simple. Advantages of PACT include tissue transparency, fluorescence preservation, and immunostaining compatibility for obtaining three-dimensional structures of mouse brain tissue. Additionally, it can enhance the penetration of antibodies in immunostaining and allows efficient immunolabeling of large volumes...
April 2018: Current Protocols in Neuroscience
Caitlin S M Cowan, Rick Richardson
Development is a time of rapid change that sets the pathway to adult functioning across all aspects of physical and mental health. Developmental studies can therefore offer insight into the unique needs of individuals at different stages of normal development as well as the etiology of various disease states. The aim of this overview is to provide an introduction to the practical implementation of developmental studies in rats and mice, with an emphasis on the study of learned fear. We first discuss how developmental factors may influence experimental outcomes for any study...
April 2018: Current Protocols in Neuroscience
Carolyn E Jones, Laura Agee, Marie-H Monfils
We describe a method of social fear transmission to a discrete auditory cue in freely behaving rats. Extending beyond traditional observer/demonstrator paradigms, rats are allowed to physically interact and integrate cues from all sensory modalities. In the protocol described in this article, "observer" rats experience social fear conditioning through a proxy cage mate that serves as a "demonstrator" during retrieval of a cued fear memory. We find that a specific auditory cue can come to elicit fear expression in an animal with no foot shock experience simply by interacting with a conspecific expressing a conditioned response in the presence of an otherwise benign stimulus...
April 2018: Current Protocols in Neuroscience
Wen-Ju Pan, Jacob Billings, Maysam Nezafati, Anzar Abbas, Shella Keilholz
Resting state functional MRI (fMRI) and functional connectivity are widely applied in humans to examine the role of brain networks in normal function and dysfunction. A similar approach can be taken in rodents, either to obtain translational measures in models of brain disorders or to more carefully examine the neurophysiological underpinnings of the networks. A protocol for resting state functional connectivity in the anesthetized rat, from animal setup to data acquisition to possible pipelines for data analysis, is described...
April 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
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