Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

Summary

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

Abstract

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.

Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.

A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions¹.

At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods^1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology⁴, cellular and molecular biology^5-8, biochemistry⁵, imaging and microscopy^4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses¹¹, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.

A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously^4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x10⁶ cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Protocol

1. Glia Dissection (~2 weeks before plating neurons)

1. To prepare for the dissection place sterile tools in 70% ethanol, add 4 mL cold dissection medium to 60 mm dishes (one dish per brain), and place 2.5% trypsin and DNase on ice to thaw (see details about surgical tools in table VI).
2. Use large scissors to decapitate 2-4 day old pups and place heads in a 100 mm sterile dish.
3. Use medium scissors to make a midline cut through the skin and pull back the skin to expose the skull. Use curved scissors to make a midline cut and two lateral cuts through the skull, without damaging the brain tissue underneath. Remove the upper skull to expose the....

Discussion

This protocol provides a method for culturing rat primary cortical neurons in the presence of glia cells, while allowing the neurons to be easily isolated for experimental analysis. The glia support development of a healthy neuronal phenotype, while also modulating neuronal responses to experimental treatments in a physiologically relevant manner. Additionally, by passaging the primary glia before culturing with neurons this cell population becomes selectively enriched in astrocytes, thereby preventing inflammatory micro.......