Current Protocols in Neuroscience最新文献

Generalization describes the transfer of conditioned responding to stimuli that perceptually differ from the original conditioned stimulus. One arena in which discriminant and generalized responding is of particular relevance is when stimuli signal the potential for harm. Aversive (fear) conditioning is a leading behavioral model for studying associative learning and memory processes related to threatening stimuli. This article describes a step-by-step protocol for studying discrimination and generalization using cued fear conditioning in rodents. Alternate conditioning paradigms, including context generalization, differential generalization, discrimination training, and safety learning, are also described. The protocol contains instructions for constructing a cued fear memory generalization gradient and methods for isolating discrete cued-from-context cued conditioned responses (i.e., “the baseline issue”). The preclinical study of generalization is highly pertinent in the context of fear learning and memory because a lack of fear discrimination (overgeneralization) likely contributes to the etiology of anxiety-related disorders and post-traumatic stress disorder. © 2020 by John Wiley & Sons, Inc.

Basic Protocol 1: Tone cued fear generalization gradient

Basic Protocol 2: Quantification of freezing

Support Protocol: Alternate conditioning paradigms

In the laboratory, memory is typically studied as a de novo experience, in which a naïve animal is exposed to a discrete learning event that is markedly different from its past experiences. Most real-world memories, however, are updates—modifications or additions—to existing memories. This is particularly true in the aging, experienced brain. To better understand memory updating, we have developed a new behavioral paradigm called the objects in updated locations (OUL) task. OUL relies on hippocampus-dependent spatial learning and has the advantage of being able to test both the original memory and the updated information in a single test session. Further, OUL relies on incidental learning that avoids unnecessary stress that might hinder the performance of aging animals. In OUL, animals first learn the location of two identical objects in a familiar context. This memory is then updated by moving one object to a new location. Finally, to assess the animals’ memory for the original and the updated information, all animals are given a test session in which they are exposed to four copies of the object: two in the original training locations, one in the updated location, and one in a novel location. By comparing exploration of the novel location to the familiar locations, we can infer whether the animal remembers the original and updated object locations. OUL is a simple but powerful task that could provide new insights into the cellular, circuit-level, and molecular mechanisms that support memory updating. © 2020 by John Wiley & Sons, Inc.

Mosaic analysis with a repressible cell marker (MARCM)–related technologies are positive genetic mosaic labeling systems that have been widely applied in studies of Drosophila brain development and neural circuit formation to identify diverse neuronal types, reconstruct neural lineages, and investigate the function of genes and molecules. Two types of MARCM-related technologies have been developed: single-colored and twin-colored. Single-colored MARCM technologies label one of two twin daughter cells in otherwise unmarked background tissues through site-specific recombination of homologous chromosomes during mitosis of progenitors. On the other hand, twin-colored genetic mosaic technologies label both twin daughter cells with two distinct colors, enabling the retrieval of useful information from both progenitor-derived cells and their subsequent clones. In this overview, we describe the principles and usage guidelines for MARCM-related technologies in order to help researchers employ these powerful genetic mosaic systems in their investigations of intricate neurobiological topics. © 2020 by John Wiley & Sons, Inc.

Combining immunological and molecular biological methods, the antibody-based proximity ligation assay (PLA) has been used for more than a decade to detect and quantify protein-protein interactions, protein modification, and protein expression in situ, including in brain tissue. However, the transfer of this technology to human brain samples requires a number of precautions due to the nature of the specimens and their specific processing. Here, we used the PLA brightfield detection technique to assess the expression of dopamine D2 receptor and adenosine A2A receptor and their proximity in human postmortem brains, and we developed a systematic random sampling method to help quantify the PLA signals. © 2019 by John Wiley & Sons, Inc.

Basic Protocol 1: Sample preparation and sectioning for PLA_BF

Basic Protocol 2: PLA_BF staining of brain tissue

Basic Protocol 3: Image acquisition and result analysis

Support Protocol: Luxol fast blue/cresyl violet staining

Cover: In Paletzki and Gerfen (http://doi.org/10.1002/cpns.84), the image shows a diagram demonstrating the process used to maintain the correct rostral-to-caudal order of tissue. The tissue is collected in a 1-in-10 series, such that each well contains every tenth tissue section and all of the tissue sections in each well remain together during all procedures.

Social transfer of fear is a potent tool facilitating response to danger in animals forming social groups. With many factors influencing the transfer—such as proximity of the animal receiving information to the donor, familiarity, proximity of danger, and species-specific coping strategies—it allows studies of neuronal correlates of a variety of behavioral responses. Since both the transfer of fear and social modulation of fear responses are impaired in many neuropsychological disorders, the models described in this article could be useful in disentangling the neuronal circuitry involved in the pathogenesis of these disorders. © 2019 by John Wiley & Sons, Inc.

Basic Protocol 1: Imminent threat in rats

Alternate Protocol 1: Imminent threat in mice

Basic Protocol 2: Remote threat in rats

Alternate Protocol 2: Remote threat in mice

Basic Protocol 3: Social modulation of fear extinction in rats

Alternate Protocol 3: Social modulation of fear extinction in mice

This unit covers some basic procedures that are common to a wide range of neuroanatomical protocols for brain tissue. Procedures are provided for preparation of unfixed fresh brain tissue as well as for perfusion fixation of animals to obtain fixed neural tissue. A variety of methods for sectioning are described, including frozen sectioning using a cryostat or microtome and sectioning with a vibratome. The choice of sectioning method depends on how the brain has been prepared and what histochemical method is to be used. A fluorescent immunohistochemical method to localize endogenous molecules as well as induced markers such as green fluorescent protein and red fluorescent protein is also provided. Additionally, three post-sectioning procedures are described: defatting of slide-mounted sections, fluorescent Nissl staining, and thionin staining of sections. Finally, support protocols are provided, describing a method for maintaining the correct order of cut tissue, whether rostral to caudal or lateral to medial; a procedure for subbing slides with gelatin, which is necessary in some protocols in order for sections to adhere to slides; and preparation of custom 3D-printed 10- or 20-well tissue plates and trays for subsequent immunostaining. Published 2019. U.S. Government.

Basic Protocol 1: Preparation of unfixed fresh-frozen brain tissue

Basic Protocol 2: Perfusion fixation

Basic Protocol 3: Cryostat sectioning of frozen brain tissue

Basic Protocol 4: Sliding-microtome sectioning of fixed brain tissue

Basic Protocol 5: Vibratome and Compresstome sectioning

Support Protocol 1: Tissue collection in a 1-in-10 series

Support Protocol 2: Preparation of gelatin-subbed microscope slides

Support Protocol 3: Custom 3D-printed 10- and 20-well tissue plates

Basic Protocol 6: Post-sectioning procedures I: Fluorescent immunohistochemical localization

Basic Protocol 7: Post-sectioning procedures II: Defatting

Basic Protocol 8: Post-sectioning procedures III: Nissl staining

Basic Protocol 9: Post-sectioning procedures IV: Thionin staining

Cover: In Suar et al. (http://doi.org/10.1002/cpns.83), the image shows typical fresh immunofluorescent staining results. The top row displays fluorescent microscopy images. Scale bars, 100 µm. The bottom row displays confocal microscopy images. Scale bars, 20 µm. (A, E) Overlay image. (B, F) Vimentin signal (fibroblasts and arachnoid trabeculae). (C, G) Collagen I signal (vasculature and arachnoid trabeculae). (D, H) DNA signal (cell nuclei).

The goal of neurogenetics is an understanding of the genetic basis of brain structure and function. Neurogenetics deals with multiple areas of investigation, including the genetic basis of neural induction, patterning, cell fate specification, neuron maturation, axonal and dendritic organization, synapse function, neural information processing, and learning and behavior. This appendix provides links to databases and other Web sites used by neurobiologists for discovery of information about genes and cellular networks involved in neural development and neuron function. Special care has been taken to curate sites involving model organisms, since great strides have been made using Drosophila and C. elegans for understanding neural development and function. Published 2019. U.S. Government.

In this article, we describe a protocol for the isolation and staining of fresh tissue of the inner rat meningeal layers, or pia–arachnoid complex (PAC). The PAC is believed to act as a mechanical damper offering a fundamental layer of protection against brain injury; however, its overall mechanical properties are still rather unexplored. In order to perform micromechanical measurements on the PAC, the tissue must be extracted and characterized while maintaining its native mechanical properties (i.e., avoiding any chemical or physical modification that could alter it). In light of this need, we developed a protocol for the immunofluorescent staining of fresh PAC tissue that does not require any fixation or permeabilization step. This approach will allow researchers to investigate important properties of the anatomy of ex vivo PAC tissue while at the same time offering a platform for the mechanical analysis of this complex material. © 2019 by John Wiley & Sons, Inc.

Basic Protocol 1: Isolation of fresh rat pia–arachnoid complex tissue

Basic Protocol 2: Fresh immunofluorescent staining of rat pia–arachnoid complex tissue

Alternate Protocol: Adhesion of pia–arachnoid complex tissue to glass slides for micromechanical characterization