Dissection, Plating, and Maintenance of Cortical Astrocyte Cultures

Survival of central nervous system neurons in culture is usually greatly improved if the neurons are plated on top of a layer of confluent astrocytes. Under these conditions, neurons attach more firmly and develop a more readily identifiable bidimensional neuritic tree than in culture conditions where primary cells are plated on top of collagen, laminin, or other cell-free substrates. Although dissociated cells from any central nervous system region will include astrocytes and other glia, these generally do not support the survival of the primary neurons as well as preplated astrocyte feeder layers. For example, plating dorsal root ganglion (DRG)/dorsal horn (DH) cocultures directly onto collagen or laminin results in a very low number of attached DH neurons, and those have stunted neurites. Furthermore, although a higher number of DRG neurons do attach without astrocytes, this attachment is weaker, as demonstrated by the tendency of those neurons (or at least their neurites) to blow off the coverslip when a local superfusion system is directed at them. Neonatal rat cortex provides abundant astrocytes that can be cultured readily. The protocol presented here selects for type I astrocytes that grow in a monolayer with contact inhibition.