Abstract
Over the last decade, the importance of astrocyte-neuron communication in neuronal development and synaptic plasticity has become increasingly clear. Since neuron-astrocyte interactions represent highly dynamic and reciprocal processes, we hypothesized that many astrocyte genes may be regulated as a consequence of their interactions with maturing neurons. In order to identify such neuron-responsive astrocyte genes in vitro, we sought to establish an expedited technique for separation of neurons from co-cultured astrocytes. Our newly established method makes use of cold jet, which exploits different adhesion characteristics of subpopulations of cells (Jirsova etal., 1997), and is rapid, performed under ice-cold conditions and avoids protease-mediated isolation of astrocytes or time-consuming centrifugation, yielding intact astrocyte mRNA with approximately 90% of neuronal RNA removed. Using this purification method, we executed genome-wide profiling in which RNA derived from astrocyte-only cultures was compared with astrocyte RNA derived from differentiating neuron-astrocyte co-cultures. Data analysis determined that many astrocytic mRNAs and biological processes are regulated by neuronal interaction. Our results validate the cold jet as an efficient method to separate astrocytes from neurons in co-culture, and reveals that neurons induce robust gene-expression changes in co-cultured astrocytes.
Highlights
During central nervous system (CNS) development, glia and neurons are targeted to their proper location in the brain and the appropriate contacts between neurons, as well as neurons and glia cells, are established
The purity of astrocyte cultures was determined by immunostaining for glial fibrillary acidic protein (GFAP) and S100β, while counterstained for all cell nuclei using Hoechst, and was determined to be higher than 98%
Since attachment of neurons to the extracellular culture environment might vary with time, cold jet removal of neurons from neuron-astrocyte co-cultures was tested at 10 and 14 day in vitro (DIV)
Summary
During central nervous system (CNS) development, glia and neurons are targeted to their proper location in the brain and the appropriate contacts between neurons, as well as neurons and glia cells, are established. While neuroscience research historically has focused on synaptic contacts between neurons, the last couple of decades it has become apparent that reciprocal neuron-glia interactions are crucial for correct brain functioning. The functionally diverse glia-neuron interactions rely on a range of both contact-dependent mechanisms as well as soluble factors (Eroglu and Barres, 2010; Giaume et al, 2010). The development of elaborate in vitro systems, using highly purified cell populations, has strongly contributed to the elucidation of the molecular effects of glia on neuronal development and function (Ullian et al, 2001; Thomson et al, 2008; Jarjour et al, 2012). Other astrocyte-secreted factors that were shown to influence synapse development are extracellular matrix molecules, such as thrombospondins and glypicans (Clarke and Barres, 2013)
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