Mini-Ruby is Rapidly Taken up by Neurons and Astrocytes in Organotypic Brain Slices

Abstract

Cholinergic neurons are intensively studied, because they degenerate in Alzheimer's disease. Although neurotracer techniques are widely used to study axonal transport, guidance, regeneration or sprouting it is not clear if cholinergic neurons can be stained by tracer techniques and studied in brain slices. The aim of the present study was to evaluate the characteristics of the neurotracer Miniruby in organotypic brain slices of the basal nucleus of Meynert (nBM), focusing on cholinergic neurons. Miniruby is a biotinylated dextran amine and is taken up very fast by a variety of cells. When 2-week old nerve growth factor-incubated brain slices of the nBM were treated with Mini-ruby crystals for 1 h, only a few (2-3%) cholinergic neurons were clearly labeled as shown by co-localization with choline acetyltransferase. The staining was found in neuN-positive neurons and microtubule associated protein-2 (MAP-2)-positive nerve fibers. A very rapid dynamic change was observed in these labeled varicosities within seconds. However, Mini-ruby was taken up also by many glutamine synthethase-positive astrocytes. At the site of Mini-ruby application an intense CD11b-positive microglial staining was evident. In conclusion, neurons and astrocytes in organotypic brain slices can be labeled very fast with the fluorescent dye Mini-ruby which undergoes dynamic processes.

Organotypic brain slice cultures: A review

“…Several applications have been reported, such as e.g. repeated multi-electrophysiological recordings and stimulations (Egert et al, 1998; Jahnsen et al, 1999; Karpiak and Plenz, 2002; Dong and Buonomano, 2005), or gene transfer techniques (Ridoux et al, 1995; Thomas et al, 1998; Murphy and Messer, 2001), retrograde tracing using fluorescent dyes (Ullrich and Humpel, 2011), or long-term live imaging (Gogolla et al, 2006). Organotypic brain slices can be analyzed using all common neurobiological methods.…”

“…The method was modified and optimized by Stoppini et al (1991), who found that organotypic brain slices survive well when cultured on semipermeable membranes. Meanwhile, this method has been used and adapted by several research groups including ours (Bergold and Casaccia-Bonnefil, 1997; Noraberg, 2004; De Simoni and Yu, 2006; Marx, 2010; Ullrich et al, 2011; Ullrich and Humpel, 2011; Shamir and Ewald, 2014). As an attractive alternative the in oculo model was developed, which allows three-dimensional tissue grafts in the anterior eye chamber to be studied.…”

Brain Slice Derived Nerve Fibers Grow along Microcontact Prints and are Stimulated by Beta-Amyloid(42)

“…Voltage-Sensitive (FluoVolt) and Calcium-Sensitive (Rhod-4) Dyes Fluorescently labeled dextrans are frequently employed in live cell imaging studies to track both anterograde and retrograde neuronal pathways, a technique firmly established in our laboratory [28]. MiniRuby (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA, D3312) is a dextran conjugate combined with the red-fluorescent dye tetramethylrhodamine.…”

Hypothalamic CSF-contacting neurons project to the rostral agranular insular cortex: an immunofluorescence and ultrastructural analysis in the rat.

“…Zhang et al, (2003) described these cells as CSF-contacting tanycytes in the ventral ventricular wall. In the literature, only glia at the site of injection of BDA was labeled with the neuronal tracer (Rajakumar et al, 1993), Mini-ruby, a member of the dextran family, labeled astrocytes in organotypic slices of the brain (Ullrich and Humpel, 2011). However, Wouterlood and Jorritsma-Byham, (1993) noted in a few cases that BDA-labelled glial or perivascular cells were identified in the injection area but the labelling of glial cells in other sites did not occur.…”