DiOlistic Labeling of Neurons in Tissue Slices: A Qualitative and Quantitative Analysis of Methodological Variations

Abstract

Fine neuronal morphology, such as dendritic spines, classically has been studied using the Golgi technique; however, Golgi staining is difficult to combine with other histological techniques. With the increasing popularity of fluorescent imaging, a number of fluorescent dyes have been developed that enable the coupling of multiple fluorescent labels in a single preparation. These fluorescent dyes include the lipophilic dialkylcarbocyanine, DiI; traditionally used for anterograde and retrograde neuronal tracing. More recently, DiI labeling has been used in combination with the Gene Gun for “DiOlistic” labeling of neurons in slice preparations. DiI sequesters itself within and diffuses laterally along the neuronal membrane, however once the cell is permeabilized, the DiI begins to leak from the cell membrane. A DiI derivative, Cell Tracker™ CM-DiI, increases dye stability and labeling half-life in permeabilized tissue, however at much greater expense. Here, the DiI and CM-DiI DiOlistic labeling techniques were tested in side-by-side experiments evaluating dye stability within dendritic architecture in medium spiny neurons of the dorsal stratum in both non-permeabilized and permeabilized tissue sections. In tissue sections that were not permeabilized, spine density in DiI labeled sections was higher than in CM-DiI labeling. In contrast, tissue sections that were permeabilized had higher spine densities in CM-DiI labeled neurons. These results suggest that for experiments involving non-permeabilized tissue, traditional DiI will suffice, however for experiments involving permeabilized tissue CM-DiI provides more consistent data. These experiments provide the first quantitative analyses of the impact of methodological permutations on neuronal labeling with DiI.

mGluR5 Positive and Negative Allosteric Modulators Differentially Affect Dendritic Spine Density and Morphology in the Prefrontal Cortex

“…Diolistic labeling of neurons was performed according to previously published procedures [14, 63, 64]. Animals were deeply anesthetized with sodium pentobarbital (150 mg/kg) via the intraperitoneal (i.p.)…”

“…The minimum and maximum values of the segment diameter for dendritic spines were set at ≥0.143 μm and ≤10.5 μm, respectively, and the fluorescence contrast threshold was set at 3 μm. These settings were adequate to distinguish all fluorescent edges which allowed for accurate representation of the shape and size of dendrites and spines analyzed [14, 63, 64, 67]. All dendrite and spine tracing were performed by an investigator blind to experimental treatment group.…”

Long-term effects of cocaine experience on neuroplasticity in the nucleus accumbens core of addiction-prone rats

“…DiOlistic methods have been described previously (Gan et al, 2000, Grutzendler et al, 2003, Moolman et al, 2004, O’Brien and Lummis, 2006, Staffend and Meisel, 2011a, b)}. Briefly, tefzel tubing (Bio-Rad, Hercules, CA) was purged with nitrogen and precoated with 10mg/ml polyvinylpyrrolidone (PVP; mol wt=360,000; Sigma-Aldrich).…”

Impact of immersion oils and mounting media on the confocal imaging of dendritic spines

“…Perfusion with 1.5% paraformaldehyde results in more complete and clearer visualization of DiI-labeled neurons when compared to perfusion with the standard 4% paraformaldehyde (Staffend and Meisel 2011b). Brains were removed and coronally blocked to allow for penetration of the post-perfusion fixative (1.5% paraformaldehyde for 1 hr) into the tissue blocks containing the striatum.…”

“…Recently, there has been greater attention towards systematically testing and quantitatively evaluating procedural variables used in preparing tissue for confocal 3D measurements of dendritic spines (Dumitriu et al 2011; Staffend and Meisel 2011b). There has been additional focus on the imaging parameters necessary to achieve optimal resolution of dendritic spines (Dumitriu et al, 2011).…”