Ballistic labeling and dynamic imaging of astrocytes in organotypic hippocampal slice cultures

University of Iowa, Iowa City, Iowa, United States
Journal of Neuroscience Methods (Impact Factor: 2.05). 02/2005; 141(1):41-53. DOI: 10.1016/j.jneumeth.2004.05.013
Source: PubMed


Protoplasmic astrocytes in mammalian CNS tissues in vivo have a highly complex 3D morphology, but in dissociated cell cultures they often assume a flattened, fibroblast-like morphology bearing only a few, simple processes. By fluorescent labeling and confocal reconstruction we show that many astrocytes in organotypic hippocampal slice cultures exhibit a more native complex cytoarchitecture. Although astrocytes at the surface of slice cultures show a reactive form with several thick glial fibrillary acidic protein (GFAP)-positive processes, astrocytes situated in deeper portions of tissue slices retain a highly complex 3D morphology with many fine spine- or veil-like protrusions. Dozens of astrocytes can be labeled in single slice cultures by gene gun-mediated ballistic delivery of gold or tungsten particles carrying cDNAs (Biolistics), lipophilic dyes (DiOlistics), or fluorescent intracellular calcium indicators (Calistics). Expression of a membrane-targeted form of eGFP (Lck-GFP) is superior to soluble eGFP for resolving fine astrocytic processes. Time-lapse confocal imaging of Lck-GFP transfected astrocytes or "calistically" labeled astrocytes show structural remodeling and calcium transients, respectively. This approach provides an in vitro system for investigating the functional architecture, development and dynamic remodeling of astrocytes and their relationships to neurons and glia in live mammalian brain tissues.

Download full-text


Available from: Michael E Dailey,
  • Source
    • "Nonviral gene delivery methods have improved enormously in recent years and can offer integration-free expression that is becoming more comparable to that of viral vectors under certain experimental conditions [10]. In targeting glial cells, nonviral genetic manipulation has been performed by physical (ballistic labelling, magnetofection), electrical (electroporation), or chemical methods (cationic polymer, cationic lipid, or calcium phosphate) [11] [12] [13] [14] [15]. Despite significant research investigation with chemical transfection formulations of cationic lipids (forming "
    [Show abstract] [Hide abstract]
    ABSTRACT: The introduction of genes into glial cells for mechanistic studies of cell function and as a therapeutic for gene delivery is an expanding field. Though viral vector based systems do exhibit good delivery efficiency and long-term production of the transgene, the need for transient gene expression, broad and rapid gene setup methodologies, and safety concerns regarding in vivo application still incentivize research into the use of nonviral gene delivery methods. In the current study, aviral gene delivery vectors based upon cationic lipid (Lipofectamine 3000) lipoplex or polyethylenimine (Viromer RED) polyplex technologies were examined in cell lines and primary glial cells for their transfection efficiencies, gene expression levels, and toxicity. The transfection efficiencies of polyplex and lipoplex agents were found to be comparable in a limited, yet similar, transfection setting, with or without serum across a number of cell types. However, differential effects on cell-specific transgene expression and reduced viability with cargo loaded polyplex were observed. Overall, our data suggests that polyplex technology could perform comparably to the market dominant lipoplex technology in transfecting various cells lines including glial cells but also stress a need for further refinement of polyplex reagents to minimize their effects on cell viability.
    01/2015; 2015(1):1-10. DOI:10.1155/2015/458624
    • "The GFP coexpressed by this vector allows transfected cells to be distinguished from non-transfected cells in real time. For controls, differentiated PC12 cells were transfected with an expression plasmid in which the CMV promoter drives the expression of a GFP protein fused to the first 26 amino acids of Lck (designated Lck-GFP) (Benediktsson et al., 2005). One day prior to transfection , differentiated PC12 cells were dissociated by trituration, gently pelleted by centrifugation (5 minutes at 410 g), and plated in NGF-containing medium on glass-bottomed dishes (WillCo Wells) coated with poly-L-ornithine (Sigma, #P-4638). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Sirt3 is a mitochondrial sirtuin whose deacetylase activity regulates facets of oxidative metabolic efficiency, anti-oxidative capacity, and intra-mitochondrial signaling. In this study, we tested whether the over-expression of a human Sirt3-myc transgene in differentiated PC12 cells, a model of sympathetic catecholaminergic neurons, would affect the sensitivity of these cells to oxidative stress or trophic withdrawal insults. Expression analysis revealed the Sirt3-myc product was expressed as a 45 kilodalton pro-form, which localized primarily within the cytosol, and a 30 kilodalton processed form that localized predominantly within mitochondria. When subjected to acute glucose deprivation or acute oxygen-glucose deprivation, differentiated PC12 cells over-expressing Sirt3-myc displayed significantly lower levels of cytotoxicity, both at the end of the insult, and at different times following media reperfusion, than cells transfected with a control plasmid. Further, Sirt3-myc over-expression also protected differentiated PC12 cells from apoptosis induced by trophic withdrawal. Collectively, these data indicate that an elevation of Sirt3 is sufficient to protect neuronal PC12 cells from cytotoxic insults, and add to the growing evidence that Sirt3 could be targeted for neuroprotective intervention.
    Brain Research 10/2014; 1587(1). DOI:10.1016/j.brainres.2014.08.066 · 2.84 Impact Factor
  • Source
    • "A main structural characteristic of astrocytes is the starshaped arborization of their stem processes. However, at the ultrastructural level, protoplasmic astrocytes have much more complex shapes consisting of thousands of very fine elaborated protrusions that make them look more like sponges than stars [14] [15] [16]. Detailed morphological descriptions and quantitative analyses provided by electron microscopy (EM), especially those based on three-dimensional (3D) reconstructions from serial ultrathin sections, have allowed the identification of astroglia and their interactions with neurons and synapses. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The function and efficacy of synaptic transmission are determined not only by the composition and activity of pre- and postsynaptic components but also by the environment in which a synapse is embedded. Glial cells constitute an important part of this environment and participate in several aspects of synaptic functions. Among the glial cell family, the roles played by astrocytes at the synaptic level are particularly important, ranging from the trophic support to the fine-tuning of transmission. Astrocytic structures are frequently observed in close association with glutamatergic synapses, providing a morphological entity for bidirectional interactions with synapses. Experimental evidence indicates that astrocytes sense neuronal activity by elevating their intracellular calcium in response to neurotransmitters and may communicate with neurons. The precise role of astrocytes in regulating synaptic properties, function, and plasticity remains however a subject of intense debate and many aspects of their interactions with neurons remain to be investigated. A particularly intriguing aspect is their ability to rapidly restructure their processes and modify their coverage of the synaptic elements. The present review summarizes some of these findings with a particular focus on the mechanisms driving this form of structural plasticity and its possible impact on synaptic structure and function.
    Neural Plasticity 01/2014; 2014(1):232105. DOI:10.1155/2014/232105 · 3.58 Impact Factor
Show more