DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

Section on Neuronal Structure, Laboratory for Integrative Neuroscience, NIH - National Institute of Health, Bethesda, MD, USA.
Journal of Visualized Experiments (Impact Factor: 1.33). 07/2010; 6(41). DOI: 10.3791/2081
Source: PubMed


DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided.
This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

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    • "ontal cortex and hippocampus ( Adalbert and Coleman , 2013 ) . Here , we showed confocal images of the DiI - labeled dendrite segments from the brain slices of both the control groups and silibinin - treated groups . Two important characteristics to look for are a sparse labeling pattern and the ability to identify individual cellular components ( Seabold et al . , 2010 ) . The cell body was medium - sized , and the dendrites were densely covered with spines . The pictures demonstrated the degree of the increase in the average number of dendritic spines on a single neuron from the mice in the silibinin - treated groups . Our results showed that the average density of dendritic spines on each neuron , w"
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    ABSTRACT: Alzheimer's disease (AD) is characterized by amyloid β (Aβ) peptide aggregation and cholinergic neurodegeneration. Therefore, in this paper, we examined silibinin, a flavonoid extracted from Silybum marianum, to determine its potential as a dual inhibitor of acetylcholinesterase (AChE) and Aβ peptide aggregation for AD treatment. To achieve this, we used molecular docking and molecular dynamics simulations to examine the affinity of silibinin with Aβ and AChE in silico. Next, we used circular dichroism and transmission electron microscopy to study the anti-Aβ aggregation capability of silibinin in vitro. Moreover, a Morris Water Maze test, enzyme-linked immunosorbent assay, immunohistochemistry, 5-bromo-2-deoxyuridine double labeling, and a gene gun experiment were performed on silibinin-treated APP/PS1 transgenic mice. In molecular dynamics simulations, silibinin interacted with Aβ and AChE to form different stable complexes. After the administration of silibinin, AChE activity and Aβ aggregations were down-regulated, and the quantity of AChE also decreased. In addition, silibinin-treated APP/PS1 transgenic mice had greater scores in the Morris Water Maze. Moreover, silibinin could increase the number of newly generated microglia, astrocytes, neurons, and neuronal precursor cells. Taken together, these data suggest that silibinin could act as a dual inhibitor of AChE and Aβ peptide aggregation, therefore suggesting a therapeutic strategy for AD treatment. Copyright © 2015 Elsevier Inc. All rights reserved.
    Neurobiology of Aging 02/2015; 36(5). DOI:10.1016/j.neurobiolaging.2015.02.002 · 5.01 Impact Factor
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    • "Typical applications of this technique include the study of neuronal morphology during development and altered development in neurological disorders (Bruce et al., 1997; Braun and Segal, 2000; Smith et al., 2009; Li et al., 2010). This dye can be applied to a variety of cell types, live or fixed tissue (Terasaki et al., 1994), as well as diverse species such as rodents, primates, and zebrafish (Gan et al., 2000; O’Brien and Lummis, 2006; Seabold et al., 2010; Arsenault and O’Brien, 2013). In slice preparations, DiI labeling is commonly known as “DiOlistic labeling,” in which beads coated with the lipophilic dye are “ballistically” ejected with a gene gun on to brain tissue (Lo et al., 1994). "
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    ABSTRACT: Analyzing cell morphology is a key component to understand neuronal function. Several staining techniques have been developed to facilitate the morphological analysis of neurons, including the use of fluorescent markers, such as DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate). DiI is a carbocyanine membrane dye that exhibits enhanced fluorescence upon insertion of its lipophilic hydrocarbon chains into the lipid membrane of cells. The high photostability and prominent fluorescence of the dye serves as an effective means of illuminating cellular architecture in individual neurons, including detailed dendritic arborizations and spines in cell culture and tissue sections. Here, we specifically optimized a simple and reliable method to fluorescently label and visualize dissociated hippocampal neurons using DiI and high-resolution confocal microscopic imaging. With high efficacy, this method accurately labels neuronal and synaptic morphology to permit quantitative analysis of dendritic spines. Accurate imaging techniques of these fine neuronal specializations are vital to the study of their morphology and can help delineate structure-function relationships in the central nervous system.
    Frontiers in Neuroanatomy 05/2014; 8:30. DOI:10.3389/fnana.2014.00030 · 3.54 Impact Factor
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    • "In the present study, we loaded the neurons of rat brain slices 111 with highly sensitive lipophilic VSDs by applying a technique of 112 biolistic delivery. The biolistic delivery was first introduced for 113 transformation of cells with genetic vectors several decades ago 114 (Sanford, 1988), and was recently shown to be applicable for 115 morphological staining of live and fixed tissues with lipophilic 116 carbocyanine and dextran-based dyes (Gan et al., 2000, 2009; 117 Lichtman et al., 2008; Morgan and Kerschensteiner, 2011; Seabold 118 et al., 2010). This technique was also used to deliver calcium sen- 119 sitive probes in brain slices and mouse brains in vivo (Kettunen 120 et al., 2002). "
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    ABSTRACT: Optical recording of membrane potential changes with fast voltage-sensitive dyes (VSDs) in neurons is one of the very few available methods for studying the generation and propagation of electrical signals to the distant compartments of excitable cells. The more lipophilic is the VSD, the better signal-to-noise ratio of the optical signal can be achieved. At present there are no effective ways to deliver water-insoluble dyes into the membranes of live cells. Here, we report a possibility to stain individual live neurons with highly lipophilic VSDs in acute brain slices using biolistic delivery. We tested four ANEP-based VSDs with different lipophilic properties and showed their ability to stain single neurons in a slice area of up to 150μm in diameter after being delivered by a biolistic apparatus. In the slices of neocortex and hippocampus, the two most lipophilic dyes, di-8-ANEPPS and di-12-ANEPPQ, showed cell-specific loading and Golgi-like staining patterns with minimal background fluorescence. Simultaneous patch-clamp and optical recording of biolistically stained neurons demonstrated a good match of optical and electrical signals both for spontaneous APs (action potentials) and stimulus-evoked events. Our results demonstrate the high efficiency of a fast and targeted method of biolistic delivery of lipophilic VSDs for optical signals recording from mammalian neurons in vitro.
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