Current Protocols in Neuroscience最新文献

This review describes several transgenic mouse models of Alzheimer's disease (AD), a devastating neurodegenerative disorder that causes progressive cognitive decline and is diagnosed postmortem by the presence of extracellular amyloid-β (Aβ) plaques and intraneuronal tau neurofibrillary tangles in the cerebral cortex. Currently there is no intervention that cures, prevents, or even slows disease progression. Its complex etiology and pathology pose significant challenges for animal model development, and there is no single model that faithfully recapitulates both the pathological aspects and behavioral phenotypes of AD. Nearly 200 transgenic rodent models of AD have been generated primarily based on mutations linked to Aβ protein misprocessing in the familial form of the disease. More recent models incorporate mutations in tau protein, as well as mutations associated with the sporadic form of the disease. The salient features, strengths, limitations, and key differentiators for the most commonly used and best characterized of these models are considered here. While the translational utility of many of these models to assess the potential of novel therapeutics is in dispute, knowledge of the different models available and a detailed understanding of their features can aid in the selection of the optimal model to explore disease mechanisms or evaluate candidate medications. We comment on the predictive utility of these models considering recent clinical trial failures and discuss trends and future directions in the development of models for AD based on the plethora of clinical data that have been generated over the last decade. © 2019 by John Wiley & Sons, Inc.

RNA localization is an important step in gene regulation. Imaging RNAs in fixed and live cells provides contextual information about RNA distribution in the cells. Here, we provide detailed protocols for performing single-molecule fluorescence in situ hybridization (smFISH). smFISH detects mRNA molecules at single-molecule resolution in fixed neuronal cells using ∼50 small oligonucleotide probes for each mRNA. The technique has been successfully applied to understand RNA localization and distribution in various biological systems, ranging from Drosophila to the mammalian nervous system. The probes are small enough to bind to structured RNAs or RNAs that are part of RNA-protein complexes, thereby accounting for ∼85% of the total RNA; this enables a level of sensitivity equivalent to that of quantitative real-time PCR, but with anatomical resolution. © 2019 by John Wiley & Sons, Inc.

The axon initial segment (AIS) is the first 20- to 60-μm segment of the axon proximal to the soma of a neuron. This highly specialized subcellular domain is the initiation site of the action potential and contains a high concentration of voltage-gated ion channels held in place by a complex nexus of scaffolding and regulatory proteins that ensure proper electrical activity of the neuron. Studies have shown that dysfunction of many AIS channels and scaffolding proteins occurs in a variety of neuropsychiatric and neurodegenerative diseases, raising the need to develop accurate methods for visualization and quantification of the AIS and its protein content in models of normal and disease conditions. In this article, we describe methods for immunolabeling AIS proteins in cultured neurons and brain slices as well as methods for quantifying protein expression and pattern distribution using fluorescent labeling of these proteins. © 2019 by John Wiley & Sons, Inc.

Cover: In Du (https://doi.org/10.1002/cpns.69), the image shows high-power micrograph of Golgi-impregnated dendrites in the mouse cerebral cortex. Note many dendritic spines of various shapes and sizes on the dendrite (A) and on all branches of a dendrite (B).

Ribosome tagging has become a very useful in vivo approach for analyzing gene expression and mRNA translation in specific cell types that are difficult and time consuming to isolate by conventional methods. The approach is based on selectively expressing a hemagglutinin A (HA)–tagged ribosomal protein in a target cell type and then using antibodies against HA to purify the polysomes and associated mRNAs from the target cell. The original approach makes use of a mouse line (RiboTag) harboring a modified allele of Rpl22 (Rpl22-HA) that is induced by the action of Cre recombinase. The Rpl22-HA gene can also be introduced into the animal by stereotaxic injection of an AAV-DIO-Rpl22-HA that is then activated in Cre-expressing cells. Both methods for tagging ribosomes facilitate the immunoprecipitation of ribosome-bound mRNAs and their analysis by qRT-PCR or RNA-Seq. This protocol will discuss the technical procedures and describe important considerations relevant to the analysis of the data. © 2019 by John Wiley & Sons, Inc.

At the ultrastructural level, axon terminals containing synaptic vesicles are clearly observed. These axon terminals (presynaptic component of a synapse) may be seen establishing contacts (synapses) with cell bodies, axons, or dendrites (postsynaptic component of a synapse). By a combination of ultrastructural analysis and immunodetection of molecules, it is possible to determine the subcellular distribution of specific cellular markers (i.e., enzymes), neurotransmitters (within synaptic vesicles), vesicular transporters (in association with vesicles), and receptors (within the presynaptic or postsynaptic component of a synapse). Here we will provide detailed protocols that facilitate the ultrastructural detection of cellular markers, receptors, and vesicular transporters. These protocols include brain ultrastructural immunodetection of one, two, or three different types of molecules prior to brain tissue processing for ultrastructural analysis (pre-embedding immunolabeling), brain molecular immunodetection after tissue processing for ultrastructural analysis (post-embedding immunolabeling), or molecular immunodetection in purified synaptic vesicles. Published 2019. This article is a US Government work and is in the public domain in the USA.

Haloperidol is a first-generation antipsychotic used in the treatment of psychoses, especially schizophrenia. This drug acts by blocking dopamine D2 receptors, reducing psychotic symptoms. Notwithstanding its benefits, haloperidol also produces undesirable impacts, in particular extrapyramidal effects such as tardive dyskinesia (TD), which limit the use of this and related drugs. TD is characterized by repetitive involuntary movements occurring after chronic exposure therapy with haloperidol. Symptoms most commonly manifest in the orofacial area and include involuntary movements, tongue protrusion, pouting lips, chewing in the absence of any object to chew, and facial grimacing. The most serious aspect of TD is that it may persist for months or years after drug withdrawal and is irreversible in some patients. This unit, aimed at facilitating the study of TD, describes methods to induce TD in rats using haloperidol, as well as procedures for evaluating the animals's TD-related symptoms. © 2019 by John Wiley & Sons, Inc.

The Golgi-Cox method has been one of the most effective techniques for studying the morphology of neuronal dendrites and dendritic spines. However, the reliability and time-consuming process of Golgi-Cox staining have been major obstacles to the widespread application of this technique. To overcome these shortcomings and to promote this invaluable technique, we developed the FD Rapid GolgiStain™ Kit based on the principle of the methods described by Ramón-Moliner in 1970 and Glaser and Van der Loos in 1981. The kit significantly improves and simplifies the Golgi-Cox technique. This kit is reliable for visualizing morphological details of neurons, allowing for analysis of various parameters of dendritic morphology—such as dendritic length and branching pattern and dendritic spine number, shape, and size—in both animal and postmortem human brains. A 40-min instructional video for tissue freezing, cryosectioning, and staining is provided. © 2019 by John Wiley & Sons, Inc.